Microstamping activated polymer surfaces

ABSTRACT

Methods of attaching a ligand to a surface are described that include contacting a surface with a substrate containing an amphiphilic comb polymer. The substrate is configured to provide a pattern of the amphiphilic comb polymer on a selected region of the surface. The substrate can be separated from the surface leaving the amphiphilic comb polymer on the selected region of the surface, thus providing a selected region of the surface having amphiphilic comb polymer on it. A ligand can then be deposited on the surface such that the selected region of the surface having the amphiphilic comb polymer is substantially free of the ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of Ser.No. 09/519,038, filed Mar. 3, 2000, now U.S. Pat. 6,444,254, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of microcontact printing.

BACKGROUND OF THE INVENTION

Micropatterning of biomolecules on surfaces has a number ofapplications, including the modulation of cell-substrate interactions inbiomaterials and tissue engineering and the fabrication of multi-analytebiosensors and genomic arrays. See Blawas, A. S. et al, Biomaterials(1998), 19, 595; Mrksich, M. and Whitesides, G. M., TIBTECH (1995) 13,228. Microcontact printing (also referred to herein as “μCP”) methodsare attractive for micropatterning of biomolecules, because of theirsimplicity and ease of use. See Kumar, A. .et al., Acc. Chem. Res. 1995,28, 219; Xia, Y. et al., Angew. Chem. Int. Ed. Engl. (1998), 37, 550. Todate, however, methods of microcontact printing have generally beenlimited to the production of patterns on self assembling monolayers(SAMs), which in turn are bound to gold or silicon surfaces. Forexample, Whitesides and coworkers have used reactive μCP to patternbiological ligands onto reactive SAMs on gold. J. Lahiri,et al.,Langmuir 1999, 15, 2055. Bernard et al. have similarly used μCP topattern different proteins onto SAMs on gold by physical adsorption. SeeBernard, A.; et al., Langmuir 1998, 14, 2225.

U.S. Pat. No. 5,512,131 to Kumar et al. proposes a method of patterninga surface in which an elastomeric stamp with a stamping substratesurface is coated with a self-assembled monolayer-forming species havinga functional group selected to bind to a surface. The stamp is thenplaced against the surface to leave a self-assembled monolayer of thespecies originally coated onto the stamp. The description of theinvention is, however, limited to the use of self-assembling monolayers.While SAMs are commonly used, the limitation of being required to usethem is disadvantageous in that SAMs generally bind only to certainmaterials such as metals (usually gold), silicon dioxide, galliumarsenide, glass, and the like. The patent fails to provide any exampleof a non-SAM species being used to bind directly to a surface, nor doesthe patent recite any examples of microcontact printing onto a materialother than gold.

While SAMs on gold are generally used for micropatterning, they havelimited utility as biomaterials. In contrast, polymers are widely usedas biomaterials. (Zdrahala, R. J., J. Biomater. Appl. (1996) 10, 309).Most previous studies on micropatterning on polymers have utilizedphotolithography. (Mooney, J. F.et al., Proc. Natl. Acad. Sci. (USA)1996, 93, 12287. Wybourne, M. N. et al., Nanotechnology 1996, 7, 302.Hengsakul, M et al., Bioconj. Chem. 1996, 7, 249. Schwarz, A.et al.,Langmuir 1998, 14, 552; Dewez, J.-L.et al., Biomaterials 1998, 19).Alternative methods have also been demonstrated by Ghosh and Crooks, whopatterned hyperbranched poly(acrylic acid) on oxidized poly(ethylene)using reactive μCP. (Ghosh, P.; Crooks, R. M. J. Am. Chem. Soc., 1999,121, 8395).

The micropatterning of biological molecules onto surfaces is animportant objective because such patterning enables, for example,control of cell-substrate interactions. (Chen, C. S.; et al., Science1997, 276, 1425. Mrksich, M.et al., Exp. Cell Res. 1997, 235, 305. Chen,C. S;et al., Whitesides, G. M.et al., Biotechnol. Prog. 1998, 14, 356).In the last decade, biomolecules have been immobilized onto the surfaceof different polymers in order to modulate their interaction with cells.(Shakesheff, K.et al., J. Biomater. Sci., Polym. Ed. 1998, 9, 507; Cima,L. G., J. Cell. Biochem. 1994, 56, 155; Massia, S. P. et al., J. Biomed.Mater. Res. 1991, 25, 223; Brandley, B. K.; et al., Anal. Biochem. 1988,172, 270. Massia, S. P. et al., Anal. Biochem. 1990, 187, 292). Morerecent studies have focused on patterning polymer surfaces withbiological ligands. Mooney, J. F.; Hunt, et al., Proc. Natl. Acad. Sci.(USA) 1996, 93, 12287. Wybourne, M. N.et al., Nanotechnology 1996, 7,302; Hengsakul, M.et al., Bioconj. Chem. 1996, 7, 249; Schwarz, A.; etal., Langmuir 1998, 14, 5526; Dewez, J.-L.et al., P. G. Biomaterials1998, 19).

Despite the foregoing, current attempts to micropattern biologicalligands onto polymer surfaces are severely limited. Most μCP methods aredone and indeed are required to be performed on gold or similar metalsurfaces. Typically, a SAM-molecule is stamped onto a gold surface tocreate a patterned SAM layer on the gold surface. See Kumar, A. .et al.,supra. In a modification of this basic method, Lahiri et al., supra,have developed a method in which a homogeneous SAM is formed on gold byincubating the gold surface in a solution of the SAM-forming molecules.Next, a stamp is used to transfer a non-SAM reactive molecule to theSAM/gold surface. The reactive molecule reacts with a reactive moleculein the SAM to form a pattern of the reactive molecule on the SAM/goldsurface. These methods are limiting because they are restricted to theuse of gold or other SAM forming surfaces, and require the use ofSAM-forming molecules. These approaches are not applicable to polymersurfaces because SAMs do not generally form on polymers. In yet anotheralternative approach (Bernard et al., supra), a stamp “inked” withprotein is used to stamp a pattern of the protein onto a polymer. Asignificant limitation of this method is that the protein is not boundto the polymer surface via a stable, covalent linkage or bond. Rather,the protein is attached to the polymer surface by physical adsorption.This approach is limiting because many molecules of interest cannot bestably bound to polymer surfaces by non-specific physical adsorption,and the patterned molecule is easily removed from the polymer surface bywater, buffers, biological fluids and the like.

In addition to the above, it has become increasingly important toattempt to control the placement of cells in an organized pattern on asubstrate for the development of cellular biosensors, biomaterials, andhigh-throughput drug screening assays. See e.g., R. Singhvi, G.Stephanopoulos, D. I. C. Wang, Biotechnology and Bioengineering 1994,43, 764, J. A. Hammarback, S. L. Palm, L. T. Furcht, P. C. Letourneau,J. Neurosci. Res. 1985, 13, 213, and K. E. Healy,B. Lom, P. E.Hockberger, Biotechnology and Bioengineering 1994, 43, 792. A potentialproblem in spatially directing cellular interactions at a biomaterialsurface is the relatively rapid adsorption of a complex layer ofproteins within a relatively short period of time of contact with serumin cell culture or upon implantation in vivo. See e.g., T. A. Horbett,J. L. Brash, ACS Sym. Ser. 1987, 343, 1.; J. D. Andrade, V. Hlady, S. I.Jeon, in Hydrophilic polymers: Advances in Chemistry Series, Vol. 248(Eds: J. E. Glass), ACS, Washington, D.C. 1996, p 51. The adsorbed layerof proteins may potentially physically obscure the micropatternedcell-adhesive ligand, or present a multitude of alternative cellularsignals, which has the ability to prevent the formation of cellularpatterns, mediated by the micropatterned cell-adhesive ligand.

One proposed approach to address this problem involves the presentationof a biochemical ligand of interest against a protein-resistant,nonfouling surface. A method to prevent nonspecific protein adsorptioninvolves the incorporation of polyethylene glycol (PEG) at the surface.A number of methods have been proposed to incorporate PEG at surfaces,including physisorption (e.g., J. H. Lee, P. Kopeckova, J. Kopecek, J.D. Andrade, Biomaterials, 1990, 11, 455; J. A. Neff, K. D. Caldwell, P.A. Tresco, J. Biomed. Mater. Res. 1998, 40, 511), chemisorption (e.g.,K. L. Prime, G. M. Whitesides, Science 1991, 25215, 1164.; K. L. Prime,G. M. Whitesides, J. Am. Chem. Soc. 1993, 115, 10714), chemical grafting(e.g., J. M Harris, in Polyethyleneglycol Chmistry; bioechnicqal andBiomedical Applications: Plenum Press, New York, 1992; K. D. Park, W. G.Kim, H. Jacobs, T. Okano, S. W. Kim, J. Biomed. Mater. Res., 1992, 26,739.; Y. C. Tseng, K. Park, J. Biomed. Mater Res. 1992, 26, 373.; M.Amiji, K. Park, J. Biomater.Sci., Polym. Ed. 1993, 4, 217),plasma-initiated grafting (e.g., M. S. Sheu, A. S. Hoffman, J. G. A.Terlingen, J. Feijen, Clin. Mater. 1993, 13, 41), and deposition (e.g.,G. P. Lopez, B. D. Ratner, C. D. Tidwell, C. L. Haycox, R. J. Rapoza, T.A. Horbett, J. Biomed. Mater. Res. 1992, 26, 415). Most of thesemethods, however, typically require multiple processing steps that areoften optimized for the surface of interest.

Thus, the successful patterning of biological ligands directly ontopolymer surfaces using reactive microstamping techniques (e.g., in whichreactions between the ligands and the polymer surfaces occur to create astable covalent bond between the two) has heretofore remained elusive.Accordingly, a need exists for a reliable method of microstampingbiological and other ligands directly and covalently onto polymersurfaces that may render the surface biologically nonfouling.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide methods of reactivemicrocontact printing (μCP) that may overcome many of the shortcomingspresented by the conventional methods described above. In a preferredembodiment, these methods enable biological ligands and proteins to bedirectly patterned onto polymers such that the resulting pattern has aspatial resolution of at least 5 μm and good reproducibility. In otherembodiments of the methods according to the present invention mayprovide spatial control of ligand presentation on the surface ofcommonly used polymeric biomaterials.

According to embodiments of the present invention, methods of attachinga ligand to a surface include contacting a surface having an amphiphiliccomb polymer present thereon, the amphiphilic comb polymer having afirst reactive moiety attached thereto, with a substrate having at leastone ligand thereon, the ligand comprising a second reactive moiety,wherein the second reactive moiety of the ligand and the first reactivemoiety of the amphiphilic comb polymer form a covalent bond; and thenseparating the substrate from the surface, thereby leaving the ligandcovalently bound to the amphiphilic comb polymer.

In other embodiments of the present invention, methods of attaching aligand to a surface include contacting a surface with a substratecontaining an amphiphilic comb polymer, wherein the substrate isconfigured to provide a pattern of the amphiphilic comb polymer on aselected region of the surface; separating the substrate from thesurface, thereby leaving the amphiphilic comb polymer on the selectedregion of the surface; and then depositing a ligand on the surface suchthat the selected region of the surface having the amphiphilic combpolymer thereon is substantially free of the ligand.

In still further embodiments, methods of attaching a ligand to a surfaceinclude depositing at least one ligand on a surface; contacting thesurface having the ligand present thereon with a substrate having anamphiphilic comb polymer thereon; and then separating the substrate fromthe surface, thereby leaving the amphiphilic comb polymer bound to theligand. The bond can be physical or chemical.

The foregoing and other aspects of the present invention are explainedin detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of surface chemical derivatization to introducecarboxylic acid groups in PET.

FIG. 2 is a schematic of the method of the present invention used tomicropattern biotin-amine onto PET. The lower left panel is a 20×magnification confocal image of Alexa™ 488-labeled streptavidin (0.1 Min HBS, pH 7.4) bound to biotin-amine, which was patterned onto PET-COOHusing techniques described herein. A line profile of fluorescenceintensity of Alexa™ 488-labeled streptavidin bound to biotin-aminepatterns created by MAPS on PET-COOH is shown on its right.

FIGS. 3A and 3B are fluorescence images of Alexa™ 488-strepatvidin boundto biotin micropatterns fabricated on PET using, as shown in FIG. 3A, anunoxidized PDMS stamp or, as shown in FIG. 3B, a PDMS stamp oxidized bya 1 min. air plasma treatment.

FIGS. 4A, 4B, 4C and 4D illustrate 10× magnification confocal images ofAlexa™ 488 labeled streptavidin patterns from a 9 mm×9 mm area,patterned with biotin-amine on PET-COOH by MAPS. A PDMS stamp with 10 μmsquare feature and an interfeature spacing of 5 μm was used to generatethese patterns. FIG. 4A is a schematic of the patterned area, showingthe location of regions from which the fluorescence images in panels 4B,4C and 4D were taken.

FIGS. 5A, 5B, 5C, 5D and FIG. 5E are (+) Time-of-Flight Secondary IonMass Spectrometry (TOF-SIMS) spectra of: PET (FIG. 5A); PET-COOH (FIG.5B); PET-COOH derivatized with biotin-amine (FIG. 5C), PET-COOHderivatized with AEE (FIG. 5D), and PET-COOH derivatized withbiotin-amine and incubated with streptavidin (FIG. 5E). Eachmodification step was confirmed by the presence of molecular ions uniquefor each molecule. P represents characteristic PET ions (M=repeat unitof PET). B represents characteristic biotin ions. A representscharacteristic AEE ions. S represents characteristic streptavidin ions.

FIG. 6 is a (−) TOF-SIMS spectrum of PET-COOH reacted with biotin-amine.P represents characteristic PET ions. B represents characteristic biotinions. F represents characteristic PFP ions. The presence of PFP speciesindicates the activating agent is not fully quenched in thederivatization reaction.

FIGS. 7A, 7B, 7C and 7D are TOF-SIMS images of a sample of biotinpatterned onto activated PET using a 40 μm stamp. The map of the CN⁻ ion(26 Da) in FIG. 7A confirms the presence of biotin created by the stamp.In FIG. 7B, the map of the C₆F₅O⁻, the PFP molecular anion (m/z 183⁻),shows that PFP is preferentially located in the unstamped regions. Themap of the molecular ion (m/z 227⁺) corresponds well with the CN⁻ map(FIG. 7C). In FIG. 7D, the map of the m/z 104 molecular ion of PETindicates that PET is preferentially exposed in the unstamped regions.Images shown in FIGS. 7A and 7B were acquired from the same area, andthe images in FIGS. 7C and 7D were acquired from different areas.

FIGS. 8A, 8B, and 8C are TOF-SIMS images of a streptavidin pattern. ThePET-COOH surface was first patterned with biotin-amine using a 10 μmstamp and subsequently incubated with streptavidin in the presence ofTween20™. FIG. 8A is the molecular map of m/z 104 ion (C₇H₄O⁺) showsthat PET is exposed in the unstamped regions. The characteristicstreptavidin ion m/z 70 ion (C₄H₈N⁻) in FIG. 8B shows that streptavidinbinds preferentially to the stamped biotin pattern. FIG. 8C shows thatresidual Tween 20™ is preferentially adsorbed to the unstamped regions.

FIGS. 9A, 9B, 9C and 9D illustrate the effect of Tween 20™ blockingagent (BA) on binding of streptavidin to a biotin micropattern. TOF-SIMSimages of micropatterned biotin on PET-COOH incubated with streptavidinwere acquired with (FIGS. 9A and 9B) and without Tween 20™ (FIGS. 9C and9D) in the protein solution: FIG. 9A and 9C m/z 104 (C₇H₄O⁺ ion from PETsurface); FIG. 9B and 9D m/z 26 (CN⁻). The square 10 μm biotin patternin images of FIG. 9A and 9B included Tween 20™ in the streptavidinbinding buffer, and show that streptavidin binds preferentially to thebiotinylated regions with low nonspecific adsorption of protein to thebackground. In contrast, the square 40 μm biotin pattern in images shownin FIGS. 9C and 9D did not include Tween 20™ in the streptavidinsolution, and show significant nonspecific adsorption of streptavidin tothe patterned surface.

FIG. 10 illustrates embodiments of delivering biological ligand to apolymeric surface having a plurality of amphiphilic comb polymersattached thereto in accordance with the present invention.

FIG. 11 illustrates embodiments of delivering biological ligand to apolymeric surface using a substrate that includes a plurality of wellsin accordance with the present invention.

FIG. 12 illustrates the chemical structure of an amphiphilicpoly(MMA/HPOEM/POEM) comb polymer.

FIG. 13A, 13B and 13C are schematic diagrams of embodiments ofmicropatterning techniques in accordance with the present invention.

FIGS. 14A and 15A are fluorescent images of protein and peptidemicropatterns on spin-cast thin films of the poly(MMA/HPOEM/POEM) combpolymer on different polymer surfaces. FIG. 14A is Alexa488-labeledstreptavidin on PET. FIG. 15A is biotin-GRGDSP-(K-TMR) peptide incubatedwith a streptavidin micropattern on PET.

FIG. 14B and 15B show line profiles of fluorescence intensity of thepatterns shown in FIGS. 14A and 15B, respectively.

FIGS. 16A and 16B illustrate phase contrast images of NIH 3T3fibroblasts aligned along the micropatterned 40 μm wide lines of thepeptide GRGDSPK fabricated on: TCPS (FIG. 16A), and PET (FIG. 16B).

FIG. 16C illustrates a spin cast comb polymer film on TCPS without apeptide micropattern is illustrated in FIG. 16C.

FIGS. 17 and FIGS. 18A, 18B and 18C are schematic diagrams ofembodiments of micropatterning techniques in accordance with the presentinvention.

FIGS. 19A and 19B illustrate the reflection image of a silicon surfacemicropatterned with an amphiphilic comb polymer as visualized by opticalmicroscopy.

FIGS. 19C is an atomic force microscopic image of a comb polymer stripepattern having a 20 μm stripe.

FIG. 19D is a line profile of the stripe shown in FIG. 19C.

FIGS. 20A, 20B and 20C and FIGS. 21A, 21B, and 21C show TOF-SIMS imagesof comb polymer patterns combined with FN.

FIG. 22A illustrates the cell repellent, “nonfouling” effect of the combpolymer spincast on tissue culture polystyrene (TCPS).

FIG. 22B illustrates that cells did not attach on the normal hydrophobicPS surface with a serum-containing medium as on comb polymer surface.

FIGS. 22C illustrates aligned cell micropatterns of fibroblasts on asurface of PS adsorbed with FN, onto which a comb polymer wasmicropatterned.

FIGS. 22D illustrates aligned cell micropatterns of fibroblasts on asurface of PET adsorbed with FN, onto which a comb polymer wasmicropatterned.

FIGS. 22E illustrates aligned cell micropatterns of fibroblasts on asurface of PMMA adsorbed with FN, onto which a comb polymer wasmicropatterned.

FIG. 22F, 22G and 22H shows micropatterns of a comb polymer printed onan FN-adsorbed surface and the resulting attachment of cells.

FIGS. 23A, 23B, 23C, 23D, 23E, 23F, 23G and 23H show time dependent cellpatterning using an amphiphilic comb polymer.

FIGS. 24A, 24B, 24C, 24D, 24E, 24F and 24G are a schematic illustrationsof embodiments micropatterning techniques using elastomeric microwellreservoirs.

FIGS. 25A and 25B are optical micrographs of PDMS molds filled with anaqueous solution. FIG. 25A illustrates square microwells and FIG. 25Billustrates microchannels.

FIGS. 26A, 27A, and 28A are micropatterns fabricated using anelastomeric microwell reservoir with biotin-NH₂/Alexa488-streptavidin(FIG. 27A), biotin-NH₂/streptavidin/biotin—GRGDSP(K-TMR) (FIG. 28A), andcovalent patterning of GRGDSP(K-TMR)-NH₂.

FIGS. 26B, 27B, and 28B are line profiles of the micropatterns in FIGS.26A, 27A, and 28A, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Polymers useful as the polymeric surfaces according to embodiments ofthe present invention may be natural polymers (e.g., biologicalpolymers) or synthetic polymers. For ease of discussion, the surfacesdescribed herein may be described as polymeric surfaces. However, othersurfaces known to those of ordinary skill in the art onto which a combpolymer can form a stable coating may be used. For example, othersurfaces that may also be used include metals, metal oxides,semiconductors, ceramics, and other composites, e.g., any material ontowhich a comb polymer can be shown to form a stable coating in water orother biologically relevant liquid, such as a cell culture medium,serum, plasma, or an exudate at an implant site. Synthetic polymers thatmay be used in the present invention include but are not limited toknown synthetic polymers such as poly(ethylene terephthalate) (PET),polystyrene (PS), polycarbonate (PC), poly(epsilon-caprolactone) (PECLor PCL), poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA),polydimethylsiloxane (PDMS), polybutadiene (PB), polyvinylalcohol (PVA),fluorinated polyacrylate (PFOA), poly(ethylene-butylene) (PEB), andpoly(styrene-acrylonitrile) (SAN). Polymers according to the presentinvention also encompass biological polymers, such as peptides,proteins, and repeating units of nucleic acid (i.e., DNA and RNA). Inany event, the term “polymer” will be defined herein as a compound ormolecule comprising at least two units of a monomer or repeating unit,as these terms are understood in the art. The term “polymer” as usedherein is also intended to encompass a homopolymer, heteropolymer,co-polymer, ter-polymer, etc., and blends, combinations and mixturesthereof.

Ligands referred to herein are preferably biological ligands, althoughnon-biological ligands such as synthetic polymers that are naturallyreactive or functionalized to be reactive with other reactive groups orfunctional groups are also encompassed by this term. Biological ligandsof the present invention include but are not limited to proteins,peptides, nucleic acids, carbohydrates, lipids, polysaccharides, andother biological molecules. Biological molecules may include, forexample, biotin, vitamins, cofactors, coenzymes, receptor agonists orantagonists, etc. The biological ligand may selectively bind variousbiological or other chemical species such as proteins, antibodies,antigens, sugars and other carbohydrates, and the like. Moreover, thebiological ligand may comprise a member of any specific or non-specificbinding pair, such as either member of the following exemplary list:antibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor,enzyme/cofactor, binding protein/substrate, carrier protein/substrate,lectin/carbohydrate, receptor/hormone, receptor/effector, complementarystrands of nucleic acid, repressor/inducer, or the like.

Non-biological ligands of the present invention include syntheticpolymers and plastics which are known in the art.

Certain embodiments of methods described according to the presentinvention are sometimes referred to herein by the acronym “MAPS,” anabbreviation for Microstamping onto an Activated Polymer Surface. Themethod involves functionalized polymer surfaces having reactive moieties(also referred to as “reactive groups” or “functional groups” herein) onthe surface of the polymer. The functionalized polymer surface iscontacted with a stamp having on its surface ligands comprising reactivemoieties that react with the reactive moieties on the polymer surface toproduce a covalent bond.

In one aspect, embodiments of the present invention provide methods ofattaching a ligand to a polymer surface. The method comprises contactinga surface having an amphiphilic comb polymer present thereon, theamphiphilic comb polymer having a first reactive moiety attachedthereto, with a substrate having at least one ligand thereon, the ligandcomprising a second reactive moiety, wherein the second reactive moietyof the ligand and the first reactive moiety of the amphiphilic combpolymer form a covalent bond; and then separating the substrate from thesurface, thereby leaving the ligand covalently bound to the amphiphiliccomb polymer. The methods described herein may be particularlyadvantageous in that in certain embodiments, the presence of theamphiphilic comb polymer has the ability to minimize or prevent thenon-specific adsorption of proteins onto a surface. Moreover, in apreferred embodiment, the polymeric surface can be modified in aone-step process in which the biological ligand is attached thereto.

In some embodiments, the contacting step comprises impressing a stamphaving at least one biological ligand present thereon, which biologicalligand comprises a second reactive moiety to the polymer surface suchthat the at least one biological ligand is covalently bound to theamphiphilic comb polymer. In these embodiments, the stamp is thesubstrate and the at least one biological ligand is attached to thesurface of the stamp. This embodiment is illustrated in greater detailin FIG. 10 with a polymeric surface 100 having a plurality ofamphiphilic comb polymers 110 attached thereto. A stamp 120 isconfigured such that it may be impressed upon the polymeric surface, andis made up of a series of protrusions 135 and wells 140. The dimensiond₁ of a protrusion 135 preferably ranges from about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, or 45microns to about 50, 55, 60, 65, 70 75, 80, 85, 90, 95 or 100 microns,and the dimension of d₂ of a well 140 preferably ranges from about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 3, 5, 10, 15, 20, 25, 30, 35,40, or 45 microns to about 50, 55, 60, 65, 70 75, 80, 85, 90, 95 or 100microns. Biological ligands 130 are present on the surface ofprotrusions 135 and are thereafter brought into contact with andcovalently bonded to the comb polymers 110 in a manner described herein.

In different embodiments, the substrate comprises at least one well andan aqueous solution which comprises the at least one biological ligandis present in the at least one well. An illustration of such embodimentsis provided in FIG. 11. As shown in FIG. 11, a substrate 150 includes aplurality of wells 155 formed therein. The width w₁ of such wells 155preferably ranges from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, or 45 microns to about 50, 55,60, 65, 70 75, 80, 85, 90, 95 or 100 microns. Each well 155 may containaqueous solution 160 having biological ligand present therein. Thesubstrate 150 is configured to impress upon or contact a polymericsurface 170 having a plurality of comb polymers 180 attached thereto,such that the aqueous solution 160 delivers biological ligand to surface170. The biological ligand in the aqueous solution 160 is then bound tosome of the plurality of comb polymers 180.

Preferably, the substrate is configured to provide a pattern of the atleast one biological ligand on the polymer surface. In an embodiment,the pattern dimensions range from about 0.1, 0.5, 1, 5, 10, 50, 100,150, or 200 μm to about 250, 300, 350, 400, 450, or 500 μm. The patterndimensions can be the same as the dimensions for the width of the wellsdescribed above. The term “pattern dimensions” refers to the dimensionsof a particular pattern formed or the spaces between the pattern. Forexample, the dimensions of a pattern of stripes are either the width ofthe stripes or the distance between the stripes and the dimensions of apattern of rectangles are the length or width of the rectangles or thedistance between the rectangles.

In other embodiments of the present invention, methods of attaching aligand to a surface include contacting a surface with a substratecontaining an amphiphilic comb polymer, wherein the substrate isconfigured to provide a pattern of the amphiphilic comb polymer on aselected region of the surface; separating the substrate from thesurface, thereby leaving the amphiphilic comb polymer on the selectedregion of the surface; and then depositing a ligand on the surface suchthat the selected region of the surface having the amphiphilic combpolymer thereon is substantially free of the ligand. Certain embodimentsare illustrated in FIG. 17. As shown in FIG. 17, a stamp such as apoly(dimethyl siloxane) (PDMS) stamp 200 includes a comb polymer 210.The stamp 200 is applied to a polymer surface 215 to which a biologicalligand 220 has been applied. The stamp 200 is separated from the polymersurface 215 and the comb polymer 210 is deposited on the biologicalligand 220 to form a patterned surface including exposed areas of thebiological ligand 240 and comb polymer 210.

In still further embodiments, a method can include depositing at leastone ligand on a surface; contacting the surface having the ligandpresent thereon with a substrate having an amphiphilic comb polymerthereon; and then separating the substrate from the surface, therebyleaving the amphiphilic comb polymer bound to the ligand. The bond canbe physical or chemical. Certain embodiments are depicted in FIGS.18A–18C. As shown in FIG. 18A a PDMS stamp 1300 includes a comb polymer1310. The stamp 1300 can be similar to the stamp 200 in FIG. 17. Thestamp 1300 is applied to a polymer surface 1315 to deposit the combpolymer 1310 onto the polymer surface 1315 when the stamp 1300 isseparated from the polymer surface 1315 as shown in FIG. 18B. Abiological ligand can then be applied to the remaining surfaces as shownin FIG. 18C to form biological ligand surfaces 1340 and surfaces havingthe comb polymer 1310 thereon.

The bond between the ligand and the comb polymer can be various bonds aswill be understood by those skilled in the art. For example, the bondbetween the ligand and the comb polymer can be a reactive bond or aphysical bond. As used herein, a reactive bond is a chemical bond suchas an ionic, covalent, mixed ionic/covalent, molecular recognition,coordination bond, or chelation bond. A physical bond is a bonddominated by secondary interactions such as hydrogen bonds, van der Waalforces, hydrophobic forces, and the like.

In certain embodiments, the ligand can have a first reactive moietyattached thereto. The amphiphilic comb polymer can comprise a secondreactive moiety. The second reactive moiety of the amphiphilic combpolymer and the first reactive moiety of the ligand can form a covalentbond.

In an alternative embodiment, a substrate 150 including a plurality ofwells 155 formed therein as depicted in FIG. 11 may be substituted forthe stamp 200 shown in FIG. 17 or the stamp 1300 shown in FIG. 18A todeposit the comb polymer. Each well 155 may contain aqueous solutionhaving comb polymer present therein. The substrate 150 is configured toimpress upon or contact the polymeric surface 215, such that the aqueoussolution 160 delivers comb polymer to the biological ligand 220 on thesurface 215. When the substrate 150 is substituted for the stamp 300 inthe embodiments of FIGS. 18A–C, the substrate 150 is configured toimpress upon or contact the polymeric surface 1315, such that theaqueous solution delivers comb polymer to surface 1315.

Amphiphilic comb polymers are known in the art and are described, forexample, in U.S. Pat. No. 6,207,749 to Mayes et al. Preferably, theamphiphilic comb-type polymers, most preferably present in the form ofcopolymers, contain a backbone formed of a hydrophobic, water-insolublepolymer and side chains formed of short, hydrophilic non-cell bindingpolymers.

The hydrophilic side chain polymer typically possesses a molecularweight of between 200 and 5000 Daltons. The hydrophobic backbone can bebiodegradable or non-biodegradable, depending on the desiredapplication. In one embodiment, it is preferred that the overallmolecular weight of the comb copolymer should be above about 10,000Daltons, more preferably above 20,000 Daltons, and more preferably stillabove 30,000 Daltons.

The comb copolymers can be prepared using various techniques. In oneembodiment, for example, the copolymer is prepared by copolymerizing ahydrophilic macromonomer which contains a polymerizable chain end with asecond hydrophobic monomer. Alternatively, a hydrophobic monomer can becopolymerized with a second monomer that includes suitable reactivegroups through which the hydrophilic side chains can be grafted to thebackbone. Alternatively, a hydrophobic monomer with a suitable reactiveside group can be polymerized and a fraction of those reactive sidegroups can be modified by grafting hydrophilic side chains. A definedpercentage of the non-cell binding side chains can be end-capped with asuitable ligand to elicit a specific cellular response.

Hydrophobic polymers used to impart biodegradable properties to thebackbones of the comb copolymers are preferably hydrolyzable under invivo conditions. Suitable biodegradable polymeric units include hydroxyacids or other biologically degradable polymers that yield degradationproducts -that are non-toxic or present as normal metabolites in thebody. Embodiments of such include, without limitation, poly(aminoacids), poly(anhydrides), poly(orthoesters), and poly(phosphoesters).Polylactones such as poly(epsilon-caprolactone),poly(delta-valerolactone), poly(gamma-butyrolactone)and poly(beta-hydroxybutyrate), for example, are also useful. Preferredpoly(hydroxy acid)s are poly(glycolic acid), poly(DL-lactic acid) andpoly(L-lactic acid), poly (DL-sebacic acid) or copolymers ofpoly(glycolic acid) poly(lactic acid), and/or poly(sebacic acid).

If desired, biodegradable regions can be constructed from monomers,oligomers or polymers using linkages susceptible to biodegradation, suchas, for example, ester, peptide, anhydride, orthoester, and phosphoesterbonds. Exemplary non-biodegradable, hydrophobic polymers that can beincorporated into the backbone of the comb copolymers include, withoutlimitation, polyalkylenes such as polyethylene and polypropylene,polychloroprene, polyvinyl ethers, polyvinyl esters such as poly(vinylacetate), polyvinyl halides such as poly(vinyl chloride), polysiloxanes,polystyrene, polyurethanes and copolymers thereof, polyacrylates, suchas poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate),poly(n-butyl(meth)acrylate), poly(isobutyl (meth)acrylate),poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referredto herein as “polyacrylates”), polyacrylamides such as poly(acrylamide),poly(methacrylamide), poly(ethyl acrylamide), poly(ethylmethacrylamide), poly(N-isopropyl acrylamide), poly(n, iso, andtert-butyl acrylamide) and copolymers and mixtures thereof. For thepurposes of the invention, these polymers include useful derivatives,including polymers having substitutions, additions of chemical groups,for example, alkyl groups, alkylene groups, hydroxylations, oxidations,and other modifications routinely made by those skilled in the art.

Preferred non-biodegradable polymers include ethylene vinyl acetate,polyacrylates, poly(chloroprene), and copolymers and mixtures thereof

The non-cell binding side chains are preferably water-soluble when notattached to the backbone, and, more preferably, are non-ionic. Suitablepolymeric side chains include those prepared from poly(ethylene glycol),poly(ethylene oxide), poly(propylene glycol), poly(propylene oxide),partially or fully hydrolyzed poly(vinyl alcohol),poly(vinylpyrrolidone), and dextran and copolymers thereof. It should beappreciated that other side chains can be employed as will be understoodby one skilled in the art. Preferably, the side chains comprisepoly(ethylene glycol), poly(ethylene oxide), poly(propylene glycol),poly(propylene oxide), or poly(acrylic acids). The hydrophilic sidechains may be intrinsically biodegradable or may be poorly biodegradableor effectively non-biodegradable in the body. In the latter two cases,the side chains should be of sufficiently low molecular weight to allowexcretion. The preferred molecular weight range is below about 5000Daltons, more preferably below 2000 Daltons, and most preferably, belowabout 1000 Daltons. When the polymer is polyethylene glycol, it ispreferred that the number of ethylene oxide monomeric units is betweenabout 4 and 20, i.e., an oligoethylene glycol side-chain.

In embodiments wherein double-bond containing monomers are used toprepare the polymer backbone, a preferred method for incorporating thehydrophilic side chains is to use a hydrophilic macromonomer with areactive double bond at one end which can be randomly incorporatedduring free radical or other addition polymerization. An example of sucha macromonomer is PEG-methacrylate. The density of the non-cell binding,hydrophilic side chains along the polymer backbone is controlled bycontrolling the relative amounts of the PEG-methacrylate or othersuitable macromonomeric unit used.

In those embodiments in which the side groups are end capped withcell-signaling ligands, appropriate functional groups, such as —NH₂,—OH, or —COOH are included on the ends of the macromonomers.

In certain embodiments described herein, monomers can be used to form apolymer backbone. The monomers can include two reactive groups, both ofwhich are reacted in order to form the polymer. For example, lactic acidincludes two reactive groups, a hydroxy group and a carboxy group.Hydroxy is the preferred reactive group. Although the ends of apolylactic acid polymer include a hydroxy group and a carboxy group,there are no reactive groups along the backbone in the final polymerchain that can be used to form a comb copolymer.

Monomers which contain one or more additional reactive groups need to beincorporated into the polymer backbone, preferably in a random fashion,in order to form the comb-type copolymers when monomers that do notinclude these reactive groups are used to prepare the polymer backbone.Examples of these types of monomers are well known to those of skill inthe art. Preferably, a suitable reactive monomer can be incorporated inthe growing polymer chain by participating in the same types of chemicalreactions as the growing polymer chain. For example, when lactide isbeing polymerized using a Lewis acid catalyst, a depsipeptide (cyclicdimer of an amino acid) can be prepared from lysine, in which theepsilon amine group is protected, for example, with a t-boc protectinggroup. The lysine is incorporated into the polymer, and the protectinggroup can be removed. The resulting amine groups are reactive withhydrophilic polymers which include leaving groups such as tosylates,tresylates, mesylates, triflates and other leaving groups well known tothose of skill in the art.

Alternatively, the reactive monomer can include a leaving group that canbe displaced with a nucleophilic group on a hydrophilic polymer. Forexample, epichlorohydrin can be used during the polymerization step. Themonomer is incorporated into the polymer backbone, and the chloridegroup is present on the backbone for subsequent reaction withnucleophiles. An example of a suitable hydrophilic polymer containing anucleophilic group is a PEG with a terminal amine group. PEG-NH₂ canreact with the chloride groups on the polymer backbone to provide adesired density of PEG-ylation on the polymer backbone. Using thechemistry described herein, along with the general knowledge of those ofskill in the art, one can prepare polymer backbones which includesuitable leaving groups or nucleophiles for subsequent couplingreactions with suitably functionalized hydrophilic polymers.

Polymer surfaces of the present invention are preferably flat or planar,but may be also be curved, cylindrical, or shaped according to theuser's needs. For example, the polymer surface may also be corrugated,rugose, concave, convex or any combination of these conformations. Thepolymer surface may have various shapes such as, but not limited to, afilm or sheet of polymer, a strand, a tubing, a sphere, a container, acapillary, a pad, a molded plastic device, or a plastic plate (e.g., atissue culture plate). The polymer surface may be on prosthetic orimplantable device on which it is desired to covalently bond certainligands, and which ligands may be capable of binding or attracting othercompounds or biological matter (e.g., cells, proteins, or otherbiological materials).

A functionalized polymer surface according to embodiments of theinvention can have at least one reactive moiety on its surface. Thepolymer surface may be functionalized by means known in the art toproduce reactive moieties on the surface of the polymer. Reactivemoieties known to those of skill in the art include, but are not limitedto, amine groups, sulfur-containing functional groups such as thiols,sulfides, disulfides, and the like; silanes and chlorosilanes;carboxylic acids; nitrites and isonitriles; and hydroxamic acids.Additional suitable reactive moieties include acid chlorides,anhydrides, sulfonyl groups, phosphoryl groups, azo, diazo and hydroxylgroups. Presently, —COOH or carboxylic acid groups are preferred.Exemplary reactive moieties may be hydrophobic, hydrophilic,amphipathic, ionic, nonionic, polar, nonpolar, halogenated, alkyl, oraryl. A non-limiting, exemplary list of such reactive moieties includes:—H, —CONH—, —CONHCO—, —NH₂, —NH—, —COOH, —COOR, —CSNH—, —NO₂ ⁻, —SO₂ ⁻,—RCOR—, —RCSR—, —RSR′, —ROR—, —PO₄ ⁻³, —SO₃ ⁻², —SO₃ ⁻, —NH_(x)R_(4−x+),—NH_(x)R_(3−x+), —COO⁻, —SOO⁻, —RSOR—, —CONR′₂, —(OCH₂CH₂)_(n)OH (wheren=1–20, preferably 1–8), —CH₃, —PO₃H⁻, -2-imidazole, —N(CH₃)₂, —NR₂,—PO₃H₂, —CN, —(CF₂)_(n)CF₃ (where n=1–20, preferably 1–8), olefins, andthe like. In this list, R′ is hydrogen or an organic group such as ahydrocarbon or fluorinated hydrocarbon, and R is an organic group suchas a hydrocarbon or fluorinated hydrocarbon. Where the reactive moietycontains more than one R, it is to be understood that each R isindependently selected. As used herein, the term “hydrocarbon” includesalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, and thelike. The hydrocarbon group may, for example, comprise methyl, propenyl,ethynyl, cyclohexyl, phenyl, tolyl, and benzyl groups. The term“fluorinated hydrocarbon” is meant to refer to fluorinated derivativesof the above-described hydrocarbon groups. Alternatively, R may be abiologically active species such as an antigen, antibody, hapten, etc.Additional reactive moieties suitable for use in the present inventionmay also be found in U.S. Pat. No. 5,079,600, issued Jan. 7, 1992.

Functionalization, as used herein, means a method comprising one or moresteps in which a reactive moiety is introduced onto the surface of thepolymer. Functionalization may occur in one, two, or even more steps,with each step generally being a chemical or thermal modification of apolymer surface, the end result of which is a surface onto which areactive group is introduced. For example, when the polymer surface isPET, the PET surface may first be hydroxylated, and then reacted withone or more compounds as known in the art to introduce carboxylic acidmoieties onto the surface. The carboxylic acid moities may then beactivated (e.g., using pentaflurophenol) in order to make the carboxylicacid group moieties reactive with other reactive moieties. Severalmethods of introducing reactive groups onto the surface of polymers areknown, including hydrolysis (Kumar, D. J.; Srivastava, H. C. J. Appl.Polym. Sci. 1987, 33, 455.; Solbrig, C. M. et al., J. Appl. Polym. Sci.:Appl. Polym. Symp. 1991, 47, 437; Búi, L. N., et al. Analyst. 1993, 118,463) and reduction (Búi, L. N.et al., Analyst. 1993, 118, 463; Chen,W.et al., Langmuir 1998, 14, 5586). Other chemical approaches that canbe used to introduce reactive groups onto the surface of the polymer arephotoinitiated graft polymerization, (Yao, Z. P.; et al., J. Appl.Polym. Sci. 1990, 41, 1459) aminolysis, (Avny, Y.et al., J. Appl. Polym.Sci. 1986, 32, 4009), the formation of a surface interpenetratingnetwork of poly(ethylene oxide), (Desai, N. P.et al., Macromolecules1992, 25, 226), chemical reaction at hydroxyl end-groups (Mougenot, P.etal., J. Macromolecules 1996, 29, 3552; Mougenot, P.; et al., J. Coll.Interfac. Anal. 1996, 177, 162), corona discharge, (Strobel, M. et al.,J. Adhes. Sci. Technol. 1992, 5, 429), reactive plasma etching, (Wang,J.et al., J. Appl. Polym. Sci., 1993, 50, 585), laser treatment(Bertrand, P.et al., Nucl. Meth. Phys. Res., Sect. B. 1987, 19–20, 887)and ion beam modification. (Arenolz, E. et al., Appl. Surf. Sci. 1993,69, 16; Ratner, B. D.et al., in Plasma Deposition, Treatment, andEtching of polymers (D'Agostino, Ed.; Academic Press, Inc.: New York,1990).

In embodiments of the present invention, the functionalized surface ofthe polymer having a reactive moiety thereon is contacted with a stampcomprising on its surface a ligand comprising a reactive moiety.Reactive moieties of the ligand may be any of the reactive moieties setforth above, as long as they are able to covalently bind to thefunctionalized polymer surface.

Stamps useful in the present invention are known in the art and may becommercially available. Generally, these stamps are produced by castinga polymeric material onto a mold having the desired pattern. Theparticular material chosen for formation of the stamp is not critical tothe present invention, but should be chosen so as to satisfy certainphysical characteristics. In a preferred embodiment, the stamp iselastomeric. Polymeric materials suitable for use in the fabrication ofthe stamp may have linear or branched backbones, and may be crosslinkedor non-crosslinked, depending upon the particular polymer and the degreeof formability desired of the stamp. A variety of elastomeric polymericmaterials are suitable for such fabrication, especially polymers of thegeneral classes of silicone polymers and epoxy polymers, with siliconeelastic polymers being preferred. Examples of silicone elastomerssuitable for use as the stamp include those formed from precursorsincluding the chlorosilanes such as methylchlorosilanes,ethylchlorosilanes, and phenylchlorosilanes, and the like. Aparticularly preferred silicone elastomer is polydimethylsiloxane(PDMS).

The stamp should also be formed such that the stamping surface comprisesa material that ligands of the present invention may adsorb to. Theinvention may be carried out using stamps as described in U.S. Pat. No.5,817,242 to Biebuyck et al. In a particularly preferred embodiment, thestamp is oxidized, and more preferably is plasma-oxidized, prior tocontact with the polymer surface.

The term “ligand,” as used herein, means any molecule or compoundcapable of forming a covalent bond with another reactive molecule orcompound. Stated another way, a ligand is a molecule that willcovalently bind to a complementary site on another structure orcompound. The site of binding may be the reactive moiety of anothercompound, e.g., a polymer. The ligands of the present inventionaccordingly comprise at least one reactive moiety (also referred toherein as a “reactive group”). The reactive moiety of the ligand may beat the physical terminus of the ligand, or at any site on the ligandavailable for forming a covalent bond. Reactive moieties will varyaccording to the particular ligand being used and the reactive group onthe functionalized surface of the polymer to which the ligand willcovalently bind. Stated another way, a ligand will comprise a reactivemoiety compatible with the ligand and capable of binding to the reactivemoiety on the surface of the functionalized polymer surface, such thatthe reactive moiety of the ligand and the reactive moiety of the polymersurface form a covalent bond. Reactive moieties of the present inventionmay be any one of the reactive moieties listed above as possiblereactive moieties for the functionalized polymer surface, with aminegroups being presently preferred. Ligands may naturally comprisereactive moieties, or reactive moieties may be attached or bound to theligand in some way.

In embodiments of the invention, the reactive moiety is connected to theligand by means of a spacer molecule. The spacer may be polar;non-polar; halogenated or, in particular, fluorinated; positivelycharged; negatively charged; or uncharged. For example, a saturated orunsaturated, linear or branched alkyl, aryl, or other hydrocarbon spacermay be used. In some embodiments of the invention, the spacer ispolyethylene glycol; in other embodiments, the spacer is an ethyleneglycol oligomer.

The ligands of the present invention are generally attached to the stampby adsorption, which adsorption techniques are known in the art.

Ligands, both biological and non-biological, may be cytophilic, that is,adapted to promote cell adhesion or cell attraction to the ligand. Cellsthat adhere to these ligands may be whole or fractionated cells. Suchligands may be extracellular matrix proteins, fibronectin, collagen,laminin, serum albumin, polygalactose, sialic acid, and various lectinbinding sugars. Ligands may also be “biophilic,” that is, may adhere orattract certain biological molecules or compounds. These ligand includeantibodies or fragments of antibodies and their antigens, cell surfacereceptors and their ligands, nucleic acid sequences and many others thatare known to those of ordinary skill in the art.

In practicing the present invention, the stamp being used may bereferred to as being “inked” by the ligands. In general, this “inking”means that the stamp comprises on its surface a plurality of ligands, bywhich is meant either more than one of a particular ligand (i.e., morethan one molecule of a ligand), or a variety of different ligands (e.g.,molecules of different species, such as a protein and a small biologicalmolecule). In other words, the stamp surface may comprise a heterogenousmixture or a homogenous sample of ligand. The stamp may be inked with asolution comprising the ligands that will be adsorbed to the stamp. Thissolution may be referred to as “the ink.” Accordingly, the inking may,for example, be accomplished by (1) contacting the stamp with a material(e.g., paper, sponge) moistened with the ink, (2) pouring the inkdirectly onto the stamp, (3) applying the ink to the stamp with a anappropriate application device (e.g., a cotton swab, brush, sprayer,syringe, etc.), or (4) dipping the stamp surface into the solution. Theink may be allowed to dry on the stamp or may be blown dry. The inkedstamp is then placed into contact with the functionalized polymersurface for a length of time sufficient for the reactive moieties of thepolymer surface and the ligand to bond covalently. The period of timefor this will of course vary with the ligands, reactive moieties andpolymers being used, but will be able to be determined by one skilled inthe art. For example, contacting the stamping surface with the surfacepolymer for a period of time of approximately 10 minutes is generallyadequate to effect sufficient transfer, but contact may be maintainedfor longer or shorter periods of time if necessary or appropriate.

Once the stamp inked with the ligand is contacted to the polymer surfacefor a time sufficient to allow the covalent bonding of the reactivemoieties of the polymer surface to the reactive moieties of the ligand,the stamp is then separated or removed from the polymer surface, leavingthe ligand covalently bound to the functionalized polymer surface. Theligands bound to the polymer surface may, if desired, be treated ormodified further by chemical or thermal treatments known in the art.Additionally, if a reactive moiety located on the ligand remains exposedand available to binding by another ligand, such an additional ligand (a“second ligand”) may be contacted to the ligand bound to the polymersurface. For example, if the ligand bound to the polymer surface isbiotin or biotin-amine, then streptavidin, known to bind to biotin, maybe contacted with the bound biotin to form a binding pair. If the ligandis a receptor, a putative receptor agonist or antagonist may becontacted to the polymer surface to determine if the agonist orantagonist compound binds to the receptor. If the ligand is an antibodybound to the polymer surface, the second ligand may be an antigen orhapten.

A polymer surface of the present invention may be stamped more than onetime according to methods of the invention. Stamps subsequent to thefirst stamp may be the first stamp “re-inked,” different stamps, stampswith different patterns thereon, and stamps comprising different ligandson the stamp surface.

Although useful in patterning methods (i.e., wherein the stamp containsone or more indentations to produce a pattern of bound ligands on thepolymer surface), it is to be understood that the stamp may not haveindentations on it. In other words, use of the stamp may create anuninterrupted “lawn” or “block” of ligands covalently bound to thesurface of the polymer.

Devices that comprise polymer surfaces microstamped by the methods ofthe present invention are thus also an aspect of the invention. As willbe apparent to those of ordinary skill in the art, the direct binding ofbiological and other ligands to polymers is important in many areas ofbiotechnology including, for example, production, storage and deliveryof pharmaceutical proteins, purification of proteins by chromatography,design of biosensors and prosthetic devices, and production of supportsfor attached tissue culture. The present methods find use in creatingdevices for adhering cells and other biological molecules into specificand predetermined positions. Accordingly, one example of a device of thepresent invention is a tissue culture plate comprising at least onesurface microstamped by the method of the present invention. Such adevice could be used in a method for culturing cells on a surface or ina medium and also for performing cytometry. Furthermore, the devicecould be used in immobilization of cells at a surface and forcontrolling the shape of a cell. Such devices are useful in a wide arrayof cellular biology applications, including cell culturing, recombinantprotein production, cytometry, toxicology, cell screening,microinjection, immobilization of cells, influencing the state ofdifferentiation of a cell including promoting differentiation, arrestingdifferentiation or causing dedifferentiation. Embodiments of the devicesof the present invention can be used to promote ordered cell-cellcontact or to bring cells close to one another, but prevent suchcontact. Other embodiments of the devices of the invention are useful inthe creation of artificial tissues for research or in vivo purposes andin connection with creating artificial organs such as artificial liverdevices. Still other embodiments of the devices may be useful inconnection with generating surfaces for prosthetic or implantabledevices.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

OVERVIEW OF EXAMPLES

In the following Examples, the present invention is illustrated bypatterning biotin onto the polymer PET. The skilled artisan willappreciate that the present method is in no way limited to the use ofbiotin and PET. PET was chosen for illustrative purposes because it is awidely used biomaterial in synthetic vascular grafts and tissue culture.(Zdrahala, R. J. J. Biomater. Appl. 1996, 10, 309. Shakesheff, K.;Cannizzaro, S.; Langer, R. J. Biomater. Sci., Polym. Ed. 1998, 9, 507.Cima, L. G. J. Cell. Biochem. 1994, 56, 155. Massia, S. P.; Hubbell, J.A. J. Biomed. Mater. Res. 1991, 25, 223). Carboxylic acid groups areutilized as the illustrative active group on the polymer surface becausethey are a convenient functional group for conjugation to a wide varietyof biomolecules. (Hermanson, G. T. Bioconjugate Techniques, AcademicPress, 1^(st) Ed., San Diego, 1996). The choice of biotin as thebiological ligand was dictated by the following reasons: (1) biotin is aprototypical small molecule biological ligand; (2) molecular recognitionbetween biotin and streptavidin (or its homologue, avidin) ischaracterized by tight noncovalent interaction (equilibriumconstant=10¹³–10¹⁵ M⁻¹), (Green, N. M. Biochem. J. 1966, 101, 774.Chilkoti, A.; Tan, P. H.; Stayton, P. S Proc. Natl. Acad. Sci., USA1995, 92, 1754. Chilkoti, A.; Stayton, P. S. J. Am. Chem. Soc. 1995,117, 10622. Wilchek, M.; Bayer, E. Avidin-Biotin Technology, Methods inEnzymology, Vol. 184, Academic Press: San Diego, 1990; Vol. 184) andtherefore permits facile patterning of streptavidin onto a biotinpattern; and (3) the homotetrameric structure of streptavidin displays222 point symmetry, (Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.;Salemme. F. R. Science 1989, 243, 85. Hendrickson, W. A.; Pahler, A.;Smith, J. L.; Satow, Y.; Merritt, E. A.; Phizackerley. R. P. Proc. Natl.Acad. Sci. (USA) 1989, 86, 2190) which positions two pairs of biotinbinding sites on opposite faces of the protein, and thereby enablesother biotinylated biomolecules (e.g., peptides, DNA, or other proteins)to be specifically immobilized onto the streptavidin pattern in asubsequent incubation step.

PS, PMMA and PET were purchased from GoodFellow Corp.of Berwyn, Pa., andwere washed with ethanol prior to use. A comb polymer was synthesized,as described previously by free radical polymerization of MMA, HPOEM(M_(n)˜526 g/mol, corresponding to n˜10 in FIG. 1) and MePOEM (M_(n)˜475g/mol, m 8.5). See e.g., D. J. Irvine, A. M. Mayes, L. G. Griffith,Biomacromolecules 2001, 2, 85. The comb polymer was carboxylated bysuccinic anhydride, as described elsewhere. The comb polymer wascharacterized by ¹H NMR in CDCl₃; 4.12 ppm (—OCH₃), 3.6–3.65 ppm(CH₂—CH₂—O—), 0.5–2 ppm (CH₂—C—(CH₃)—) and 3.39 ppm (—OH). Thecomposition of the terpolymer was 61 wt % MMA, 21 wt % HPOEM, and 18 wt% MPOEM using the peaks at 4.12 ppm and 3.39 ppm for quantification. Thenumber average molecular weight (MW) of the comb polymer (M_(n)) was˜25,000 Da with a polydispersity of ˜2.7, as measured by gel permeationchromatography using PS calibration standards.

After synthesis, the hydroxyl-functionalized comb polymer wascarboxylated by reaction with succinic anhydride in solution. Thin filmsof the poly(MMA/HPOEM/MPOEM) comb polymer were prepared by spin-coating1% (w/v) water/methanol (20/80 v/v) solutions of the comb polymer ondifferent surfaces at 2000 rpm and then drying the films at roomtemperature for 24 h. The spin-cast films were activated by immersion inan aqueous solution of 1-ethyl-3-(dimethylamino)propylcarbodiimide(EDAC, 0.1 M) and N-hydroxysuccinimide (NHS, 0.2 M) for 30 min. Thesamples were then rinsed with deionized water, dried under a stream ofnitrogen, and used immediately thereafter.

The thickness of the comb polymer film, spincast onto a silicon waferwas measured on a home-built single wavelength ellipsometer. See e.g.,Z-P. Yang, W. Frey, T. Oliver, A. Chilkoti, Langmuir 2000, 16, 1751. Thesessile water contact angles of the comb polymer films on the differentpolymer surfaces were measured on a Ráme-Hart goniometer (100-00,Mountain Lakes, N.J.) using deionized, distilled water. X-rayphotoelectron spectroscopy was performed on an SSX-100 instrument(Surface Science Labs, Mountain View, Calif.) at a take-off angle of35°, as described elsewhere. See e.g., Z-P. Yang, A. Chilkoti, Adv.Mater. 2000, 12, 413 and Z-P. Yang, A. M. Belu, A. Liebmann-Vinson, H.Sugg, A. Chilkoti, Langmuir 2000, 16, 7482.

The fabrication of elastomeric stamps with micrometer-size relieffeatures has been described previously, as has the use of MAPS to μCPEZ-Link™ biotin-PEO-LC-amine ((+)-biotinyl-3,6,9-trioxaundecanediamine)(biotin-amine) onto polymers. See e.g., Z-P. Yang, A. M. Belu, A.Liebmann-Vinson, H. Sugg, A. Chilkoti, Langmuir 2000, 16, 7482. Aftermicropatterning biotin-amine, the micropatterned surfaces were incubatedwith 0.1 μM Alexa488-labeled streptavidin or with unlabeled streptavidinin HEPES buffered saline (HBS, pH 7.4) containing 0.02% (v/v) Tween 20for 1 h. The streptavidin patterns were subsequently incubated with 0.1μM biotin-LC-Gly-Arg-Gly-Asp-Ser-Pro-Lys (biotin-GRGDSPK) in the samebuffer for 1 h or with biotin-GRGDSP(K-TMR).

NIH 3T3 cells were grown in DMEM (Gibco BRL) supplemented with 10% fetalbovine serum (FBS) (Gibco BRL), 100 units/ml penicillin, 100 mg/mlstreptomycin, and 7.5 mM HEPES at 37° C. in 5% CO₂. Cells were plated onthe peptide micropatterned slides or controls at a density of 1×10⁶cells/ml in DMEM supplemented with 10% serum. Cells were incubated at37° C. for either 3 h or 24 h, gently rinsed with culture media toremove loosely adherent cells, and imaged under phase contrast optics.

Example 1 Materials and Methods: Surface Derivatization andMicropatterning

Poly(ethylene terephthalate) films (PET, Melinex® 442/300, Dupont) werecleaned in hexane and acetone and dried under nitrogen. The cleaned PETfilms were hydroxylated by immersion in 18.5% (v/v) formaldehyde/1 Macetic acid for 4 h. at room temperature. (Massia, S. P.; Hubbell, J. A.Ann. N.Y. Acad. Sci.-Biomed. Engr. 1990, 589, 261). Subsequently, thefilms were reacted with 1 M bromoacetic acid/2 M NaOH overnight, toconvert the hydroxyl groups to carboxylic acid on the PET surface(PET-COOH). (Lofas, S.; Johnsson., B. J. Chem. Soc., Chem. Commun. 1990,1526). The PET films were activated by immersion in an ethanol solutionof 1-ethyl-3-(dimethylamino)propylcarbodiimide (EDAC, 0.1 M) andpentafluorophenol (PFP, 0.2 M) for 15 min. (Adamczyk, M.; Fishpaugh, J.R.; Mattingly, P. G. Tetrahedron Lett. 1995, 36, 8345. Kovacs, J.;Mayers, G. L.; Johnson, R. H.; Cover, R. E.; Ghatak, U. R. J. Org. Chem.1970, 35, 1810).

The masters used to cast the poly(dimethylsiloxane) (PDMS) stamps werefabricated on polished Si wafers using AZ P4620 photoresist (Clariant,Inc.), which was spin coated to a thickness of ˜5 microns and processedby contact photolithography. Elastomeric stamps were fabricated bycasting PDMS against the photoresist on silicon masters with featuresizes of 10 μm or 40 μm squares, (Wilbur, J. L.; Kumar, A.; Kim, E.;Whitesides, G. M. Adv. Mater. 1994, 6, 600) and were subsequentlyoxidized in an air plasma (150 mtorr, 40 W, 1 min.) in a plasma reactor(Plasmod™, March Instruments Inc., Concord, Cailf.), prior to use.

The ligand (+)-biotinyl-3-6,9,-trioxaundecanediamine (Pierce, hereafterreferred to as biotin-amine) was printed by contacting a plasma-oxidizedPDMS stamp, inked with 10 mM biotin-amine in ethanol, with the activatedPET-COOH surface for 10 min. Flat, plasma oxidized PDMS stamps were usedto print biotin-amine for spectroscopic analysis by XPS and TOF-SIMS.Unreacted pentafluorophenyl esters were inactivated by reaction with2-(2-aminoethoxy)ethanol (AEE, 10 mM, 0.1 M sodium bicarbonate, pH 8.3)for 20 min. The samples were cleaned with ethanol in an ultrasonic bathfor 5 min., rinsed with distilled water, and dried, prior tospectroscopic analysis.

After printing biotin-amine on PET-COOH with a PDMS stamp, the surfacewas incubated with 0.1 μM Alexa™ 488-labeled streptavidin in HEPESbuffered saline (HBS, pH 7.4) containing 0.1% (w/v) BSA and 0.02% (v/v)Tween 20 detergent for one hour.

Example 2 Summary of Methods of Analysis of Micropatterning

Because no single analytical technique is capable of elucidating thesurface chemistry of micropatterned, derivatized polymers, we haveinterrogated each step in MAPS by a suite of complementary analyticaltechniques. The primary analytical technique that we used,time-of-flight secondary ion mass spectrometry (TOF-SIMS) is one of thefew, currently available surface analysis techniques that enablesspatially-resolved molecular characterization of micropatternedsurfaces. This is because TOF-SIMS provides a mass spectrum of the top10–30 Å with high mass resolution and submicron lateral resolution. (VanVaeck, L.; Adriaens, A.; Gijbels, R. Mass Spectrom. Rev. 1999, 18, 1.Benninghoven, A., Angew. Chem. Intl. Ed. 1994, 33, 1023. Pacholski, M.L., Winograd, N. Chem. Rev., 1999, 99, 2977. Briggs, D. In PolymerSurface Characterization by XPS and SIMS. Characterization of SolidPolymers; S. J. Spells, Ed. Chapman & Hall: London, 1994; p. 312).

In concert, spectroscopic studies of functionalized polymer surfaces byX-ray photoelectron spectroscopy (XPS) provides the elementalcomposition (Briggs, D. In Polymer Surface Characterization by XPS andSIMS. Characterization of Solid Polymers; S. J. Spells, Ed. Chapman &Hall: London, 1994; p. 312. Swingle II, R. S. CRC Crit. Rev. Anal Chem.1975, 5, 267. Carlson, T. A. Photoelectron and Auger Spectroscopy;Plenum Press: New York, 1975. Andrade, J. D. In X-ray PhotoelectronSpectroscopy (XPS). Surface and Interfacial Aspects of BiomedicalPolymers. 1. Surface Chemistry and Physics J. D. Andrade, Ed.; PlenumPress: New York, 1985; p. 105. Briggs, D. and Seah, M. P. PracticalSurface Analysis, John Wiley & Sons: Chichester, 1983) as well as theconcentration of functional groups from high resolution core levelspectra (Ratner, B. D.; Castner D. G. Coll.Surf B: Biointerfaces, 1994,2, 333. Clark, D. T.; Harrison, A. J. Poly. Sci., Polym. Chem. Ed.,1981, 19, 1945–1955. Clark, D. T.; Thomas H. R. J. Poly. Sci.: Poly.Chem. Ed., 1978, 16, 791–820. Dilks, A. In X-ray PhotoelectronSpectroscopy for the Investigation of polymeric Materials. ElectronSpectroscopy—Theory, Techniques and Applications. Brundle, C. R Baker,A. D. Eds.; Academic Press: London, 1981, p. 277) and by complementarychemical derivatization methods. (Chilkoti, A.; Ratner, B. D. In SurfaceCharacterization of Advancedpolymers, Sabbatini, L., Zambonin, P. G.,Eds.; VCH: Weinheim, 1993; p. 221). We also used fluorescence microscopybecause it enables optical imaging of fluorophore-labeled proteins onmicropatterned polymers with good contrast and lateral resolution of afew microns. Accordingly, confocal fluorescence microscopy and TOF-SIMSimaging was used to examine the formation of streptavidin micropatternson the polymer surface described in Example 1.

Example 3 Materials and Methods: X-ray Photoelectron Spectroscopy

XPS analysis was carried out on an SSX-100 spectrometer (Surface ScienceIncorporated, Mountain View, Calif.), equipped with a monochromatizedA1K_(α) X-ray source, a hemispherical electron analyzer, and a lowenergy electron flood gun for charge compensation of insulators. Sampleswere typically introduced into a preparation chamber, which wasmaintained at a pressure of 10⁻⁴ torr, and then transferred into theanalysis chamber, which was typically maintained at 10⁻⁸ torr. Thesamples were analyzed at either 15° or 55° take-off angle, defined asthe angle between the sample plane and the hemispherical analyzer. Thetypical X-ray spot size was ˜600 μm. Survey scan spectra were acquiredfrom 0–1000 eV for elemental composition, and high-resolution spectra ofthe C_(1s) core level were acquired from 278–300 eV.

Example 4 Materials and Methods: Time-of-flight Secondary Ion MassSpectrometry

TOF-SIMS spectra and images were obtained on a TRIFT II TOF-SIMSinstrument (Physical Electronics, Eden Prairie Minn.). A mass-filtered⁶⁹Ga⁺ liquid-metal primary ion gun was used with a current of ˜600 pA.For spectral acquisition, the gun was operated at 15 keV and a pulsewidth of 12 ns. For imaging, the gun was operated at 25 keV and a pulsewidth of 30 ns. Details of the spectrometer are described elsewhere.(Schueler, B. Microsc. Microanal. Microstruct. 1992, 3, 119–139). Thedata was acquired over a mass range of m/z 0–1500. The data wascollected using an ion dose below the static SIMS limit of 10¹³ions/cm². A low energy electron beam was used for charge compensation.

Example 5 Materials and Methods: Fluorescence Microscopy

Fluorescence microscopy of Alexa™ 488 labeled streptavidin patterns wasperformed on a BioRad MRC 1000 confocal microscope (BioRad MicroscienceLtd., Hemel Hempstead, U.K.) with a 10× or 20× objective. The confocalmicroscope was operated at 10% power level and with a detector gain of1500 V.

Example 6 Results and Discussion: Surface Derivatization of PET

PET was derivatized in two steps to introduce carboxylic acid groups atthe surface, as shown in FIG. 1. The first step, the hydroxymethylationof the aromatic ring in PET introduced a benzylic hydroxyl group withinthe PET repeat unit, (Massia, S. P.; Hubbell, J. A. Ann. N.Y. Acad.Sci.-Biomed. Engr. 1990, 589, 261) which was then converted to acarboxyl group by reaction with bromoacetic acid. (Lofas, S.; Johnsson.,B. J. Chem. Soc., Chem. Commun. 1990,1526). After the introduction ofcarboxylic acid groups on the surface of PET (the carboxyl derivatizedPET surface is termed PET-COOH), the carboxylic acid groups were thenactivated by reaction with pentafluorophenyl (PFP) (FIG. 2). We chosePFP to activate the carboxylic acid groups in PET-COOH, because previousreports suggest that pentafluorephenyl esters are significantly morereactive than the more commonly used N-hydroxysuccinimide ester.(Adamczyk, M.; Fishpaugh, J. R.; Mattingly, P. G. Tetrahedron Lett.1995, 36, 8345. Kovacs, J.; Mayers, G. L.; Johnson, R. H.; Cover, R. E.;Ghatak, U. R. J. Org. Chem. 1970, 35, 1810) The activated PET surfacewas then patterned with biotin-amine by spatially-resolved reagenttransfer using a PDMS stamp inked with the ligand (FIG. 2). Unreactedesters were quenched by reaction with AEE.

Example 7 Results and Discussion: XPS Characterization

The carboxylation of PET was confirmed by XPS in combination withchemical derivatization with PFP. (Chilkoti, A.; Ratner, B. D. InSurface Characterization of Advanced polymers, Sabbatini, L., Zambonin,P. G., Eds.; VCH: Weinheim, 1993; p. 221). No fluorine was observed inunmodified PET after exposure to PFP/EDAC (Table 1). In contrast, 3–4(atomic) %F was measured for PET-COOH derivatized with PFP. Theseresults suggest that PET was successfully functionalized with COOHgroups. The functionalization of PET with COOH groups proceededhomogeneously within the top 50 Å of the surface because XPS analyses attwo different take off angles, 15° and 55° gave experimentallyindistinguishable results. Furthermore, the measured F/C ratio forPFP-derivatized PET-COOH indicates that the carboxylic acidconcentration in PET-COOH is ˜21% of the theoretical maximum, which wascalculated with the following assumptions: (1) 100% carboxylation of PETwithin the XPS sampling depth via the reaction scheme shown in FIG. 1,and (2) 100% derivatization of the carboxyl groups in PET-COOH byPFP/EDAC. With the above assumptions, the XPS results indicate that ˜1COOH group was introduced every 5 repeat units of PET.

TABLE I Measured and calculated atomic ratios of PFP-derivatizedPET-COOH and biotin- derivatized PET-COOH. Measured ¹Calculated SampleDerivatization N/C O/C F/C N/C O/C F/C PET — 0 0.31 0 0 0.40 0 PET 0.1 MPFP/0.2 M EDAC 0 0.32 0 0 0.40 0 PET-COOH 0.1 M PFP/0.2 M EDAC 0 0.330.06 0 0.37 0.26 PET-biotin Biotin-amine 0.023 0.33 0 0.13 0.36 0(solution) PET-biotin Biotin-amine 0.027 0.34 0 0.13 0.36 0 (flat stamp)¹The calculated atomic ratios assume the following: (1) thehydroxylation of PET proceeds specifically by hydroxymethylation of thearomatic ring in PET as shown in the reaction scheme in FIG. 1 with 100%yield; (2) all subsequent derivatization reactions also proceed with100% yield throughout the entire sampling depth of XPS.

XPS of PET-COOH, derivatized with biotin-amine using a flat,plasma-oxidized PDMS stamp inked with ligand, provided direct evidencefor the reaction of COOH groups on the surface of PET-COOH withbiotin-amine. The N/C ratio of 0.023 for PET-COOH derivatized withbiotin-amine using a flat PDMS stamp compares favorably with the N/Cratio of 0.027, obtained for derivatization of PET-COOH withbiotin-amine from solution. In contrast, no nitrogen was detected on thesurface of unmodified PET. These atomic ratios are ˜19% of thetheoretical maximum of 0.12. Upon correcting for the experimentallydetermined concentration of available COOH groups, these results alsosuggest that reaction of the available COOH groups present in PET-COOHwith biotin-amine, after activation with PFP/EDAC, proceeded tocompletion.

The XPS C_(1s) spectra of biotin-derivatized PET-COOH and native PETalso corroborate these results. The C_(1s) spectra of PET, acquired atvarious stages of the multistep functionalization procedure, were fitwith the following criteria: (1) all spectra were corrected for samplecharging using the CH_(x) component in the resolved spectra at 284.6 eVas reference. Additional peaks for the oxygen-containing functionalities(Clark, D. T.; Harrison, A. J. Poly. Sci., Polym. Chem. Ed., 1981,19,1945–1955. Clark, D. T.; Thomas H. R. J. Poly. Sci.: Poly. Chem. Ed.,1978, 16, 791–820. Dilks, A. In X-ray Photoelectron Spectroscopy for theInvestigation of Polymeric Materials. Electron Spectroscopy—Theory,Techniques and Applications. Brundle, C. R Baker, A. D. Eds.; AcademicPress: London, 1981, p. 277) were incorporated in the peak fit: theseinclude C—O—H/R (286.2 eV), COOR (288.6 eV) and a π→π* shakeup satelliteat 291.6 ev.(Gardella, J. A.; Ferguson, S. A.; Chin, R. L. Appl.Spectrosc. 1986, 40, 224). Full widths at half maximum of the componentpeaks in the spectral envelope were constrained to 1.2–1.4 eV duringcurve fitting, which is consistent with the energy resolution of theSSX-100 spectrometer.

The C_(1s) spectra of PET PET-OH, and PET COOH (Table II) werequalitatively very similar, which is due to the low level offunctionalization of PET, and the high concentration of COOR groups thatare present in native PET, which mask the incorporation of carboxylgroups in PET-COOH. The spectrum of native PET (and PET-OH and PET-COOH)were fit with four peaks, which were assigned to CH_(x), C—O—R, COOR anda π→π* shakeup satellite. The peak area ratio of 3.3:1:0.9 for theCH_(x):C—O—R:COOR species, determined by curve fitting of the C_(1s)spectrum of PET is close to the stoichiometric 3:1:1 ratio of PET.

TABLE II Resolved XPS high resolution C_(1s) spectra. Details of thepeak fit are in the text. % CH_(x) % C—N/C—S % C—O—R % COOR %π −> π*Sample (284.6 eV) (285.4 eV) (286.2 eV) (288.6 eV) (291.6 eV) PET 62.7 —18.7 16.7 1.9 PET-OH 61.7 18.8 16.5 2.0 PET-COOH 62 18.9 16.5 2.5PET-biotin 48.6 12.6 18.6 16.4 1.9 (flat stamp)

The reaction of biotin-amine with PET-COOH clearly alters the C_(1s)(Table II). Curve fitting of the spectrum of biotin-derivatized PET-COOHby the above criteria required the inclusion of a new peak, centered at285.4 eV. This peak was assigned to the C—N and C—S species in biotin.(Beamson, G.; Briggs, D. High resolution XPS of organic polymers, JohnWiley: Chichester, 1992). Assuming homogeneous functionalization withinthe XPS sampling depth, the (C—S+C—N):CH_(x) ratio of 0.23 for PET-COOHderivatized with amine-terminated using a flat stamp inked with theligand suggests that ˜1 biotin molecule is introduced into every 5repeat units in PET, which corroborate the results obtained from XPSelemental analysis.

Example 8 Results and Discussion: Fluorescence Microscopy

The spatial distribution of streptavidin on the micropatterned biotin onPET-COOH was examined by incubating the patterned surface with Alexa-488labeled streptavidin. A 20× magnification confocal image of Alexa™488-labeled streptavidin (FIG. 2, lower left panel) shows thatstreptavidin is spatially-localized on the periodic, 40 μm×40 μm biotinmicropattern printed by MAPS on PET-COOH. The average contrast ratio ofthe protein pattern in this image is 250:1 (FIG. 2, line intensityprofile), and clearly demonstrates the successful localization ofstreptavidin on the biotin pattern, mediated by molecular recognitionbetween the protein and immobilized ligand, as well as suppression ofstreptavidin adsorption on the unstamped regions, due to the presence ofBSA and Tween 20™.

In contrast, the fluorescence intensity of unmodified PET, which wassimilarly stamped with a plasma-oxidized PDMS stamp inked withbiotin-amine, followed by incubation with Alexa™ 488-labeledstreptavidin showed an average contrast ratio between the patternedregion and background of ˜40:1 (results not shown). These resultssuggest that the covalent incorporation of biotin into PET-COOH providesa six fold higher concentration of immobilized streptavidin at thesurface as compared to adsorption of biotin. There are at least twopossible reasons for the low, albeit significant streptavidin adsorptionon the control, unmodified PET stamped with biotin-amine: (1)streptavidin bound to adsorbed biotin, which was incompletely desorbedduring the rinsing procedure that preceded incubation with Alexa™488-labeled streptavidin; (2) the different surface chemistry betweenstamped and unstamped regions, due to the presence of residual biotinand PDMS transferred from the stamp, resulted in greater adsorption ofstreptavidin to the regions that were in contact with the PDMS stamp ascompared to the background.

The quality of the biotin micropattern printed by MAPS was stronglyaffected by the surface energy of the PDMS stamp. Untreated PDMS stampsproduced poor quality patterns, as shown by fluorescence microscopy ofthe binding of Alexa-labeled streptavidin (FIG. 3A). In contrast, a 1min. air plasma treatment substantially improved the transfer of ligandto the surface (FIG. 3B), presumably because the increasedhydrophilicity of the plasma-oxidized PDMS surface enabled completewetting of the surface of the PDMS stamp by the ligand solution. Thisresult is consistent with that of Lahiri et al.(supra) for reactive μCPof biotin on SAMs on gold.

We also examined the reproducibility of the biotin micropattern, bystamping a large (9 mm×9 mm) area of PET-COOH with biotin-amine using astamp with 10 μm square features and an interfeature spacing of 5 μm.The patterns were incubated with Alexa 488-labeled streptavidin andvisualized by fluorescence microscopy. FIG. 4, a composite 10× image,obtained from different regions of the surface, shows the uniformity ofthe pattern over the entire, stamped region. The loss of featureresolution and intensity, observed at the edge of the stamped regions,is probably caused by edge effects due to inhomogeneous distribution ofthe applied stress on the PDMS stamp and slight curvature of thesurface. The stability of the streptavidin pattern was also examined;after fluorescence imaging, the sample was stored in HBS (pH 7.4)containing Alexa™ 488-labeled streptavidin (0.1 μM) for a week, and thenexamined again by fluorescence microscopy. No differences were observedeither in the total intensity of the patterned regions with time or inthe contrast between patterned regions and background (results notshown).

Example 9 Results and Discussion: TOF-SIMS Spectroscopy

In order to monitor each step of the functionalization of PET by MAPS,it was necessary to first identify secondary ions that are unique toeach step of the derivatization procedure. Samples were prepared foreach derivatization step and analyzed by TOF-SIMS. The positive ionspectrum of native PET shows characteristic peaks for PET at m/z 76,104, 149, 193 [M+H]⁺ (M=repeat unit of PET), 237, 341, 385 [2M+H]⁺, 429,577 [3M+H]⁺ and 769 [4M+H]⁺ (FIG. 5A). (The Static SIMS Library, Version2, SurfaceSpectra Limited, Manchester, UK, 1999). The positive ionspectrum of PET-COOH is qualitatively similar to that of PET (FIG. 5B);the series of molecular secondary cations, characteristic of PET arealso observed in the TOF-SIMS spectrum of this sample. The peaks at m/z45 (C₂H₅O⁺) and m/z 65 (C₅H₅ ⁺), however, display increased intensityrelative to unmodified PET. Furthermore, the intensity of molecular ionsderived from PET increased by between five- and ten-fold for PET-COOHcompared to native PET. This increase in intensity was observed both inpositive (FIG. 5B) and negative ion mode (results not shown), suggestingan increased concentration of PET oligomers on the surface of themodified polymers.(Briggs, D. Surf. Interface Anal. 1986, 8, 133–136).We believe that the low MW PET oligomers are created by hydrolytic chaincleavage of PET, which also creates hydroxyl and carboxylic acidfunctionalities at the new chain ends. Therefore, the scheme shown inFIG. 1 is only approximate, and it is likely that a substantial fractionof reactive groups arise from side reactions such as hydrolytic cleavageof the PET backbone.

PET-COOH surfaces were reacted with biotin-amine by conformal contact ofa flat PDMS stamp, inked with the reagent, with the surface or byreaction from solution. TOF-SIMS provided evidence for the reaction ofbiotin with the COOH groups. FIG. 5C shows the TOF-SIMS positive ionspectrum PET-COOH reacted with biotin-amine from solution, where newpeaks at m/z 44 (C₂H₄N⁺) and 58 (C₃H₈N⁺) are observed. Molecular ions oflow intensity at m/z 227 (C₁₀H₁₅O₂N₂S⁺) and 270 (C₁₂H₂₀O₂N₃S⁺) are alsoobserved. (The Static SIMS Library, Version 2, SurfaceSpectra Limited,Manchester, UK, 1999. Briggs, D. Surf. Interface Anal. 1986, 8,133–136). These results strongly suggest the covalent reaction ofbiotin-amine with PET-COOH, because these peaks were not observed onnative PET or PET-COOH, and biotin-amine was the only nitrogencontaining species in this multi-step derivatization procedure. Further,the new peaks observed at m/z 26 (CN⁻) and 42 (CNO⁻) in the negative ionspectrum also indicate the introduction of a nitrogen-containing moiety(FIG. 6).

After derivatizing PET-COOH with biotin-amine using a patterned stamp inMAPS, the unpatterned regions were quenched with AEE. Therefore, it wasnecessary to analyze a control sample of AEE-modified PET-COOH toidentify characteristic TOF-SIMS peaks for AEE. The positive ionspectrum is dominated by peaks that are characteristic of PET (FIG. 5D).Compared to the spectra of PET-COOH, the peaks at m/z 44 (C₂H₄N⁺), 58(C₃H₈N⁺), 26 (CN⁻) and 42 (CNO⁻) (negative ions results are not shown)display significantly greater intensity. Unique peaks for AEE were notobserved, compared to biotin-derivatized PET-COOH. The importantdistinction between the spectrum of PET-COOH derivatized with AEEcompared to biotin-amine is the presence of the molecular biotin species(m/z 227⁺ and 270⁺) in the positive ion spectrum (FIG. 5C) and thegreater intensity of m/z 26⁻ and 42⁻ in the negative ion spectrum ofbiotin-derivatized PET-COOH (FIG. 6).

The negative ion TOF-SIMS spectrum of PET-COOH reacted with biotin-aminealso shows evidence of PFP, which was used to convert carboxylic acidgroups to reactive pentafluorophenyl esters (FIG. 6). The spectrumcontains significant peaks at m/z 19 (F⁻) and 183 (C₆F₅O⁻), which is theintact parent ion of PFP. The presence of these ions suggest that thereaction of the pentafluorophenyl ester with AEE and subsequenthydrolysis of unreacted pentafluorophenyl ester in the unpatterned,background region only proceeded partially to completion.

In the final step of MAPS, the biotin-derivatized PET was incubated withAlexa488-labeled streptavidin to enable protein micropatterning viamolecular recognition between biotin and streptavidin. The TOF-SIMSspectrum of this sample exhibited unique peaks for streptavidin atm/z=70 (C₄H₈N⁺) and 130 (C₉H₈N⁺) in the positive ion mode (FIG. 5E), andat m/z=46 (NO₂ ⁻) and 62 (NO₃ ⁻) in the negative ion mode (results notshown). Attribution of these ions to streptavidin was confirmed byTOF-SIMS of an adsorbed monolayer of Alexa 488 labeled streptavidin onPET (results not shown).

Example 10 Results and Discussion: TOF-SIMS Imaging

The imaging mode of TOF-SIMS was used to analyze the patterned samplesand monitor the distribution of characteristic molecular species.Patterned biotin samples were rinsed in an ultrasonic bath of ethanol,prior to analysis, to reduce PDMS contamination to background level.

TOF-SIMS imaging of cleaned samples enabled spatial mapping ofbiotin-amine, patterned onto activated PET-COOH, using a PDMS stamp with40 μm square features. The square regions of biotin were best observedby mapping the distribution of CN⁻ in the negative ion mode. The imageclearly indicates the biotin ligand is spatially localized in the 40 μmsquare contact regions (FIG. 7A). The CN⁻ map displays significantintensity in the background, which arises from AEE, the reagent used topentafluorophenyl ester groups not functionalized with biotin-amine. Thevisible contrast in the image is a consequence of the higher intensityof this peak from the biotinylated regions as compared to the backgroundregions functionalized with AEE, and demonstrates that even thedifference in peak intensity of an ion created from two different parentmolecules on the surface is sufficient to spatially map the surfacechemistry.

Although its intensity is very low, the peak at m/z 227⁺, the parentmolecular peak of biotin, can also be used to unequivocally map thedistribution of biotin. Because the surface is destroyed over time bycollision of the primary ion beam with the surface in TOF-SIMS, it islikely that the biotin molecule most likely fragmented beforesignificant signal-to-noise could be obtained in the imaging mode ofTOF-SIMS. The image of m/z 227⁺, nevertheless, even with poor signal tonoise, demonstrates that biotin is localized in the 40 μm square regions(FIG. 7C).

PFP was used to activate the entire PET-COOH surface before stampingwith biotin-amine. FIG. 7B shows the image of the parent molecular anionof PFP (m/z 183⁻) after patterning PFP derivatized PET-COOH withbiotin-amine and quenching with AEE. The peak at m/z 183 is localizedsolely to the regions where biotin is absent, which suggests thatreaction of PFP with biotin and AEE, and subsequent hydrolysis in bufferproceeds to completion in the patterned regions but is incomplete in thebackground.

FIG. 7D shows the spatial distribution of m/z 104 (C₇H₄O⁺), which isunique to PET, and shows that the intensity of this ion is highest inthe regions that do not contain biotin. Because PET is the surface, itis believed that the observed contrast is a consequence of the shallowsampling depth of TOF-SIMS for molecular ions, so that biotin moleculesin the patterned regions mask PET. In contrast, the PET signal isstronger from the background, because PFP and AEE are smaller moleculesthan biotin and may have a lower surface coverage.

The patterned biotin samples were also analyzed by imaging TOF-SIMSafter incubation with streptavidin (FIG. 8). The image of m/z 70, whichis unique to streptavidin, shows the spatial localization ofstreptavidin and reveals that the streptavidin binds selectively to the10 μm square patterned biotin regions (FIG. 8B). In contrast, the imageof m/z 104 for PET, shows higher intensity for regions of PET surfacethat were not in contact with the PDMS stamp (FIG. 8A). The two imagesshow a contrast inversion and demonstrate the successful patterning ofPET with streptavidin.

Streptavidin was incubated in the presence of Tween 20™, a blockingagent composed of polyoxyethylene sorbitan monostearate. TOF-SIMSclearly shows the presence of Tween 20™ by the characteristic peaks atm/z 227, 255 and 283 in the positive ion mode (FIG. 8C). These peaksrepresent the series of myristic, palmitic and stearic fatty acids, andare sidechains of the sorbitan molecule. (Beamson, G.; Briggs, D. Highresolution XPS of organic polymers, John Wiley: Chichester, 1992). TheTOF-SIMS image of this series of peaks shows that Tween 20™ ispreferentially located in the background region. This localization ofTween 20™ also explains the high, 250:1 contrast observed forstreptavidin in fluorescence microscopy. The high contrast of thefluorescence images can be attributed both to the selective binding ofstreptavidin to patterned biotin, as well as the preferential adsorptionof the surfactant, Tween 20, to the background. In comparison, TOF-SIMSimages of biotin-derivatized PET-COOH incubated with streptavidinwithout the addition of the Tween 20™, showed poor spatial resolution ofprotein-containing peaks. This is clearly seen upon comparing the m/z 26map (CN⁻) for a streptavidin pattern incubated with (FIG. 9B) andwithout Tween 20 (FIG. 9D). Similarly, the contrast inversion ofrepresentative secondary ions from the PET surface (m/z 104⁺) (FIG. 9A),which is a consequence of the preferential binding of streptavidin tothe biotin micropattern and the ˜1–2 nm sampling depth of TOF-SIMS, isalso substantially reduced when the blocking agent is not included (FIG.9C). These results clearly confirm that Tween 20 significantly reducesnonspecific adsorption of streptavidin to the background, unstampedregions.

Overall, the present MAPS invention finds advantage in that isapplicable to a wide variety of polymers that are amenable to surfacemodification, and thus is useful for micron scale patterning of smallmolecule ligands, peptides, and protein onto polymer surfaces forbiomaterial and biotechnological application. Micropatterning ofreactive ligands on derivatized polymer surfaces with a spatialresolution of at least 5 μM, high contrast and good reproducibility.MAPS will find particular use in the spatially-resolved immobilizationof biomolecules that are difficult to stably adsorb onto polymers, suchas small biological ligands.

Example 11 Comb Polymer Synthesis

An amphiphilic comb polymer was formed on a substrate surface accordingto the following procedure. In particular, a randompoly(MMA/HPOEM/MePOEM) terpolymer is formed by free radicalcopolymerization of the three monomers. FIG. 12 illustrates the chemicalstructure of an amphiphilic poly(MMA/HPOEM/POEM) comb polymer. NMRshowed that the comb polymer contained 61 weight percent MMA, 21 weightpercent HPOEM and 18 weight percent MPEOM, corresponding to a molarratio of 16:1:1. See e.g., D. J. Irvine, A. M. Mayes, L. G. Griffith,Biomacromolecules 2001, 2, 85. After derivatization of the terminal OHgroups in HPOEM with succinic anhydride, a small fraction (i.e., lessthan 5%) of the terminal OH groups were converted to COOH groups asobserved by the appearance of a new resonance at around 10 ppm. Seee.g., R. F. Storey, T. P. Hickey, J. Polym. Sci., Part A: Polym. Chem.1993, 31, 1825.

Example 12 Functionalized Surface Formation

The COOH-derivatized comb polymer was spincast onto a PS, tissue culturePS (TCPS), PMMA, and PET. Such polymers were selected as the candidatesurface in this example, because they are often used as biomaterials andin tissue culture. See e.g., B. D. Ratner, in Biomaterials science: anintroduction to materials in medicine; Academic Press, San Diego,Calif., 1996. It should be appreciated that surfaces formed from othermaterials can be employed in accordance with the teachings of thepresent invention.

Prior to micropatterning with MAPS, the spincast films werecharacterized by ellipsometry, atomic force microscopy (AFM), watercontact angle measurements and X-ray photoelectron spectroscopy (XPS).The thickness of a spincast film from a 1 percent (w/v) solution of thepolymer onto a silicon wafer was determined to be approximately 50 mn,and this thickness did not appreciably change upon prolonged exposure ofthe surface to water for about one month. The surfaces were uniformlycovered with comb polymer, as observed by contact mode AFM. The rootmean square roughness obtained by AFM was less than about 5 nm. Thechange in water contact angle after modification of PS and PMMA with thecomb polymer was believed to be statistically significant (p less than0.0001, unpaired t-test) (See Table 1). Spincasting the comb polymeronto PET did not significantly, if at all, change the water contactangle of the surface. Although not intending to be bound by theory, itis believed that this is due to the similar wettability of the twopolymers.

The formation of a stable film of the comb polymer was confirmed by XPS.Upon spincasting the comb polymer on PS, the XPS O/C ratio changed fromabout zero to 0.41. The experimental O/C ratio compares favorably withthe bulk O/C ratio of the comb polymer of 0.45, as determined by NMR.Again, not intending to be bound by theory, this suggests that theaverage surface composition of the comb polymer within the top 50 Å ofthe surface does not deviate substantially from the bulk composition.The C_(1s) spectrum of PS showed new peaks at 286.5 eV (C—O—R), 288.5(COOR), and the disappearance of the peak at 292 eV (π→π* shakeupsatellite) upon coating PS with the amphiphilic comb polymer.

For each surface, the water contact angle was also measured as afunction of immersion time of the film in water. In this example, it wasobserved a decrease in the contact angle upon immersion of the combpolymer-modified surfaces for 24 h in water for all three surfaces (seeTable III).

TABLE III Surface characterization of spin-cast films of the poly(MMA/HPOEM/MePOEM) comb polymer on different polymer surfaces. WaterContact angle (°)^([a]) XPS Analysis Sample t = 0 h t = 24 h At % C At %O O/C PS 88 100 0 0 PS-CP^([b]) 80 69 71.1 28.9 0.41 PET 72 71.0 27.10.38 PET-CP 74 70 73.2 26.8 0.37 PMMA 75 75.3 24.7 0.33 PMMA-CP 75 6772.6 27.4 0.38 Comb Polymer^([c]) 69.2 30.8 0.45 ^([a])Standarddeviation was typically 3° (n ≧ 6). ^([b])Spincast comb polymers wereimmersed in water for 24 h. ^([c])Theoretical values for a comb polymerwith a stoichiometry of 61 wt. % PMMA, 21 wt. % HPOEM, and 18 wt. %MePOEM.

A potentially important consequence of the reorientation of the surfaceof the comb polymer in water is that the PEG chains are believed to beoptimally positioned at the interface to be capable of performing twoimportant functions, namely confer protein resistance to the surface andfavorably present cell-specific ligands that are covalently attached tothe ends of the oligoethylene glycol chains during the micropatterningprocess.

Example 13 Protein Patterning Via MAPS

With reference to FIGS. 13A–C, a carboxylated comb polymer 210 describedin Example 10 was first spin-cast onto PS, TCPS, PMMA and PET. Afterspin-casting the comb polymer 210 onto each surface, the COOH groups inthe comb polymer 210 shown in FIG. 13A were converted toN-hydroxysuccinimide (NHS) esters to activate the surface as shown inFIG. 13B. An oxidized poly(dimethyl siloxane) (PDMS) stamp 200presenting micrometer-sized relief features was inked with anamine-linked biotin derivative (biotin-amine) 250 and brought intoconformal contact with the “activated” surface, resulting in thecovalent attachment of the biotin derivative 250 in the regions of thesurface that were in contact with the stamp 200. This procedure was usedto create periodic 40 μm wide stripes of biotin on the surface as shownin FIG. 13C that were separated by the same distance. The surface wasthen washed with PBS, pH 8.6 to hydrolyze residual NHS esters that didnot come into conformal contact with biotin-amine during stamping. Next,the surface was incubated with a solution of Alexa488-labeledstreptavidin for fluorescence microscopy, or with unlabeled streptavidinfor subsequent cell attachment studies.

FIG. 14A illustrates the formation of spatially resolved patterns offluorescently-labeled streptavidin on a biotin micropattern fabricatedon the PEG comb polymer-modified PET. Although the fluorescenceintensity in the patterned regions was believed to be lower thanprevious results using MAPS on other polymers, the nonspecificadsorption of streptavidin in the unpatterned background regions of thecomb polymer was believed to be low (see FIG. 14B) resulting in asignal-to-noise ratio (S/N) ranging from 4–6 for the different surfaces.See e.g., Z-P. Yang, A. Chilkoti, Adv. Mater. 2000, 12, 413 and Z-P.Yang, A. M. Belu, A. Liebmann-Vinson, H. Sugg, A. Chilkoti, Langmuir2000, 16, 7482.

Example 14 Presentation of Cell-Adhesive RGD Peptide on Amphiphilic CombPolymer

Streptavidin patterns were incubated with biotin-GRGDSPK. FIG. 15A showsa peptide pattern created using a fluoresecent analog,biotin-GRGDSP(K-tetramethyl rhodamine) on PET. The signal to noise (S/N)of the peptide micropatterns ranged from about 5 to about 6 on thedifferent surfaces (FIG. 15B)

The peptide micropatterns were seeded with NIH 3T3 fibroblasts from 10%serum, and the attachment and spreading of cells on the micropatternedsurfaces and controls was assessed by optical microscopy as a functionof time. By 24 h, spatially well-resolved patterns of the fibroblastswere observed on polymer surfaces (TCPS and PET) that weremicropatterned with the cell adhesive GRGDSPK peptide (FIGS. 16A and16B). In contrast, when cells were seeded onto the comb polymer filmthat had not been micropatterned with the cell-adhesive peptide, by 3 h,the cells had detached from the surface (FIG. 16C), clearlydemonstrating the lack of cell-surface attachment. Micropatterns of acontrol, GRGESP peptide on the comb polymer did not enable the formationof cellular patterns, though some cells attached to the surface, butexhibited a round morphology, indicating their inability to spread onthe surface (results not shown). These results clearly demonstrate thatthe presentation of a cell-adhesive RGD peptide on the amphiphilic combpolymer enables cells to be patterned in the presence of ECM proteins onthe surfaces of different polymeric biomaterials, mediated by thespatially-resolved presentation of the biochemical ligand.

Example 15 Cell Micropatterning Using an Amphiphilic Comb Polymer

FIGS. 17 and 18A–C show various embodiments to create highly resolvedmicropatterns of comb polymer on various surfaces. As shown in FIG. 17,a biological ligand such as fibronectin (FN) 220 can be absorbed on thepolymer surface 100. A PDMS stamp 200 can be used to microcontact-printcomb polymer 210 on the surface 200. Alternatively, as shown in FIGS.18A–C, micropatterning of comb polymer 1310 on the surface 1315 may beperformed using a PDMS stamp 1300. After the comb polymer 1310 has beendeposited on surface 1315 as shown in FIG. 18B, FN can be incubated onthe remaining exposed surface 1315 to form an exposed bioligand 1340that can comprise FN. Such methods were highly reliable and reproducibleover the entire patterning areas. Subsequently, the comb micropatterns1310 were incubated with cells to observe the preferential attachment ofcells on the FN surface (bioligand 1340).

In Example 15, a comb polymer to be micropatterned was synthesized byfree radical polymerization of methyl methacrylate, poly(ethyleneglycol) methacrylate (M_(n)˜526 g/mol) and methyl ether methacrylate(M_(n)˜475 g/mol). See Banerjee, P., Irvine, D. J., Mayes, A. M.,Griffith, L. G. Polymer latexes for cell-resistant and cell-interactivesurfaces. J. Biomed. Mater. Res. 50, 331–339 (2000); Irvine, D. J.,Mayes, A. M., Griffith, L. G. Nanoscale clustering of RGD peptides atsurfaces using comb polymers. Synthesis and characterization of combthin films. Biomacromolecules 2, 85–94 (2001). The comb polymer wascarboxylated by succinic anhydride. The comb polymer was characterizedby ¹H NMR in CDCl₃; 4.12 ppm (—OCH₃), 3.6–3.65 ppm (CH₂—CH₂—O—), 0.5–2ppm (CH₂—C—(CH₃)—) and 3.39 ppm (—OH). The composition of the terpolymerwas 61 wt % MMA, 21 wt % HPOEM, and 18 wt % POEM using the peaks at 4.12ppm and 3.39 ppm for quantification. The number average molecular weight(MW) of the comb polymer (M_(n)) was ˜25,000 Da with a polydispersity of˜2.7, as measured by gel permeation chromatography using polystyrene(PS) calibration standards.

Polymethamethylacrylate (PMMA), poly(ethylene teretphthalate) (PET) andPS were purchased from GoodFellow Corp., and were washed with ethanolprior to use. For the surfaces coated with fibronectin (FN), thesurfaces were incubated with 20 μg/ml FN solution in PBS for 1 h.Conformal Micropatterning of comb polymer was performed using anoxidized poly(dimethyl siloxane) (PDMS) stamp presentingmicrometer-sized negative features. The stamp was inked with a 1% (w/v)comb polymer solution in 50/50 (v/v) H₂O/ethanol mixture and broughtinto conformal contact with either unmodified surfaces or FN-adsorbedsurfaces, resulting in the adsorption of the comb polymer in the regionsof the surface that were in contact with the stamp according to thesteps shown in FIGS. 17 and 18A–C. Thus, periodic 20 or 40 μm widestripes, squares and circles of micropatterns were formed on the surfacethat were separated by 20 or 40 μm. The comb micropatterns on theuntreated surfaces were incubated with 20 μg/ml FN solution, and thenwashed with PBS solution. Atomic force microscpoy (AFM) and x-rayphotoelectron spectroscopy (XPS) were performed to measure the thicknessor to confirm the stability of a comb polymer layer stored in water fora month.

Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) spectra andion images were obtained on a TOF-SIMS instrument (TOF-SIMS IV, ION-TOF,Münster, Germany). A 25-keV monoisotopic ⁶⁹Ga⁺ primary ion beamgenerated by a Ga⁺ gun were used. “Bunched mode” was used to achievehighest mass resolution (m/Δm≈10,000) in the mass spectra. The typicaltarget current of the primary Ga⁺ beam in the bunched mode for TOF-SIMSwas 3 pA with a pre-bunched pulse width of 20 ns. The raster area of theGa⁺ ion gun was 384×384 μm² and the raster resolution was 128×128pixels, in order to attempt to match the pixel size with the Ga⁺ ionbeam spot size (i.e. the Ga⁺ ion beam spot size was approximately384÷128≈3 μm), thereby optimizing both spatial resolution and data ratesfor these gun conditions. All primary Ga⁺ ion fluences were below thedamage threshold of 1×10¹³ ions cm⁻² for static SIMS.

NIH 3T3 cells were grown in RPMI1640 (Gibco BRL) supplemented with 10%fetal bovine serum (FBS) (Gibco BRL), 100 units/ml penicillin, 100 mg/mlstreptomycin, and 7.5 mM HEPES at 37° C. in 5% CO₂. Cells were plated onthe comb polymer micropatterned samples or controls at a density of1×10⁵ cells/ml in RMPI1640 supplemented with 10% serum. Cells wereincubated at 37° C. for 24 h, gently rinsed with culture media to removeloosely adherent cells, and imaged under phase contrast optics for amonth.

Referring to the comb polymer regions depicted in FIGS. 19A–B, thereflection image of the silicon surface micropatterned with comb polymerwas taken visualized by optical microscopy. The resolution of thepatterned comb polymer in FIGS. 19A–B was comparable to microcontactprinting of thiols or biological molecules such as biotin andstreptavidin. See Yang, Z-P., Chilkoti, A. Microstamping of a biologicalligand onto an activated polymer surface. Adv. Mater. 12, 413–417(2000). The bright area in the image is the silicon surface that has notcontacted the PDMS surface. The dark area of the image is the combpolymer surface transferred onto the silicon surface from the PDMS stampinked with a solution of the comb polymer. (FIGS. 19A–B) The AFM heightimage of the comb stripe pattern from FIG. 19B is shown in FIG. 19C. Thesurface profile of the comb stripe pattern from FIG. 19B is shown inFIG. 19D. The thickness of the comb polymer layer was about 150 nm asmeasured by atomic force microscopy (AFM). The stability of the comblayer in water was investigated with AFM and water contact anglemeasurement. The comb layer was stable at least for about a monthwithout any deformation or desorption in aqueous solution. X-rayphotoelectron spectroscopy (XPS) also confirmed the existence of a thincomb layer after prolonged storage in water.

FN was selected for the selective attachment of cells because the RGD(arg-gly-asp) sequence of FN is a ligand for the integrin superfamily ofcell-surface receptors present on a number of different mammalian cells,and has been implicated in the adhesion and spreading of these cells onthe extracellular matrix (ECM). See Pierschbacher, M. D., Ruoslahti, E.Cell attachment activity of fibronectin can be duplicated by smallsynthetic fragments of the molecule. Nature 309, 30–33 (1984). FN waseither adsorbed on the surfaces prior to microcontact-printing of a combpolymer on it or incubated with surfaces that had beenmicrocontact-printed with a comb polymer as shown in FIGS. 17 and 18A–C.The selective adsorption of FN on the surface was proved using time offlight-secondary ion mass spectroscopy (TOF-SIMS). TOF-SIMS was selectedbecause it is a surface analytical technique with attributes that areuseful in analysis of the subtle chemical changes on the surface.TOF-SIMS can provide high-resolution (m/Δm˜10,000) mass spectra of thesurface with extremely high detection sensitivity. Because the secondaryions are formed within the top 1 to 2 monolayers of the solid surface,TOF-SIMS can be surface sensitive, which may be useful for analyzing theadsorption of ligands on the surface. TOF-SIMS ion imaging can alsoprovide spatial distributions of mass-resolved secondary ions emittedfrom the surface with submicron lateral resolution.

Positive and negative TOF-SIMS spectra and images were obtained with thesurfaces patterned with comb polymer. In order to characterize thesurface, TOF-SIMS measurements were performed on polystyrene samples.The main contributions to the positive spectrum of PS arise fromunsaturated hydrocarbon peaks. Among those, peaks at m/z=77 (C₆H₅ ⁺) andm/z=91 (C₇H₇ ⁺) correspond to aromatic ions and are highlycharacteristic of the presence of styrene rings. (results not shown) Atthe same time, the presence of the comb polymer was followed byTOF-SIMS. The positive peaks at m/z=31, 44 and the negative peaks atm/z=12, 31 were attributed respectively to CH₃O⁺, C₂H₄O⁺ and O⁻, CH₃O⁻and are unique to the comb polymer. From the positive reference spectrumof FN on PS, TOF-SIMS detected nitrogen-containing peaks at m/z=18 (NH₄⁺), m/z=30 (CH₄N⁺), and m/z=44 (C₂H₆N⁺) that are characteristic of FN.In the negative spectrum of FN, the most characteristic peaks in theconsidered mass range were at m/z=16 (O⁻), m/z=26 (CN⁻), m/z=32 (S⁻) andm/z=42 (CNO⁻).

The imaging mode of TOF-SIMS was used to analyze the patterned samplesand monitor the spatial distribution of characteristic molecularspecies. FIGS. 20A–C and 21A–C show TOF-SIMS images of the comb polymerpatterns combined with FN. CH₃O⁺ and CH₃O⁻ ions were selected forimaging the patterns of comb polymer containing oligo(ethyl glycol)molecules. Each feature is approximately 40 μm, and the spacing betweenfeatures is the same. Nitrogen-containing ions are characteristic of thepresence of FN as described above. In case of FN-adsorbed polystyrene,CNO⁻ ion from FN showed a high contrast image because of the existenceof a comb polymer layer covering FN. In addition, the CN⁻ ion image froma FN-coated PS showed a high contrast while the same ion image ofFN-incubated comb polymer patterns showed a lower contrast. The contrastimage also appeared with the ion of C₂H₆N⁺. It resulted from thenonspecific adsorption of FN on comb polymer patterns during incubation,however, the adsorption was minimized by antifouling effect of EG chainsin comb polymer.

From control experiments, it was determined that the comb polymersurface is antifouling, and the methods can be a route for the cellattachment on various surfaces. To verify this, several polymermaterials were selected which are widely used as a biomaterial; tissueculture polystyrene (TCPS), poly(ethylene terephthalate) (PET),polymethylmethacrylate (PMMA) and polystyrene (PS). See Ratner, B. D.Biomaterials science: an introduction to materials in medicine; AcademicPress: San Diego, Calif., 1996. The comb polymer spincast on TCPS wasantifouling to cells (FIG. 22A). Even after the adsorption of FN on thecomb polymer surface, just a few cells attached on the surface due tothe strong antifouling effect of flexible oligo(ethylene glycol) chains.(FIG. 22B) Compared with the results described above, comb micropatternson the FN-adsorbed PS created well-aligned cell micropattern indicatingthe affinity of FN to cells. (FIGS. 22C–E)

Without wishing to be bound by a single theory, the preferentialattachment of cells on the space between comb polymer patterns may becaused by two reasons: 1) FN adsorbed on the region of a comb polymermay have a low surface density. That is, the nonfouling effect of a combpolymer minimizes the adsorption of FN on the surface and keeps thesurface density of ligands lower on the comb polymer than is needed foranchoring cells on the surface. 2) The comb polymer may directly repelcells. The possible attachment of cells due to the low concentration ofFN on the comb polymer region can be obviated by first coating thesurface with the ligand, e.g. by adsorbing FN on the surface followed bythe formation of micropatterns of a comb polymer that are microcontactprinted or deposited from microwells on the FN-coated surface (FIGS.22F–H).

Cell patterning using an amphiphilic comb polymer showed that reliableand reproducible cell patterns can be created even after long-termincubation of the surfaces in serum without any significant cellspreading outside the patterned areas of FN. (FIGS. 23A–H) The timedependence of cell patterns was investigated for a month by opticalmicroscopy under phase contrast optics. For the square patterns, thecell patterns were stable at least for two weeks, and a few connectionsof cells between adjacent features were observed after that time. (FIGS.23A) However, some cell patterns kept their shape even after a month.For the stripe patterns, the cell patterns were still reasonably welldefined after a month. (FIGS. 23B) The results in FIGS. 23A–H may becompared with bovine serum albumin (BSA), with which the cell patternscould be kept their original dimension for 1 h and no more patternsafter 4 h incubation, and ethylene glycol thiols, whose application waslimited on gold. Ostuni, E., Kane, R., Chen, C. S., Ingber, D. E.,Whitesides, G. M. Patterning mammalian cells using elastomericmembranes. Langmuir 16, 7811–7819 (2000). Chen, C. S.; Mrksich, M.;Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric control of celllife and death. Science 1997, 276, 1425–1428. Chen, C. S.; Mrksich, M.;Huang, S.; Whitesides, G. M.; Ingber, D. E. Micropatterned surfaces forcontrol of cell shape, position, and function. Biotechnol. Prog. 1998,14, 356–363. Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.;Whitesides, G. M. Exp. Cell Res. 1997, 235, 305–313. Therefore, a combpolymer can be as a physico-chemical barrier to cell spreading inMicropatterning on various surfaces.

Example 16 Microwell Patterning on a Substrate Surface

With reference to FIGS. 24A–G, elastomeric microwell reservoirs wereused to pattern a biolmoecule of interest on a substrate surface. Asshown in FIG. 24A, poly(dimethylsiloxane) (PDMS) stamp 300 with positiverelief features was cast from a silicon master 310 with negative relieffeatures resulting in a microwell 320 (FIGS. 24B and 24C). SiloxanePolymers; Clarson, S. J., Semlyen, J. A., Eds; Prentice Hall: Englewood,N.J., 1993. As shown in FIGS. 24B and 24C, the microwells 320 weremolded from the elastomeric PDMS stamp 300. Xu, B.; Arias, F.; Brittain,S. T.; Zhao, X. M.; Grybowski, B.; Torquato, S.; Whitesides, G. M. Adv.Mater. 1999, 11, 1186–1189. Alternatively, the microwells 320 can bedirectly fabricated by casting PDMS against a silicon or photoresistmaster with positive relief features. (not shown) This process is morelaborious than directly casting from a microfabricated silicon master.However, the use of the silicon master is reduced, which may reduce thecost. As shown in FIG. 24D, the microwell 320 can then be filled with asolution 330. The solution 330 can contain a ligand.

As shown in FIGS. 25A–B, several shapes of microwells and channels maybe molded from elastomeric PDMS masters. Each mold may providehigh-resolution microstructures with feature sizes as small as 30 μm.FIG. 25A is an optical micrograph of a PDMS mold with square microwellsfilled with aqueous solution, and FIG. 25B is an optical micrographdepicting microchannels.

A PDMS mold was oxidized with an air plasma (80 W, 1 min, Plasmod, MarchInstruments) to make the surface uniformly hydrophilic (not shown). Thetop surface of the microwell was contact-printed with hexadecanethiol(HDT) using a flat, oxidized PDMS stamp (not shown) and allowed to dry,which selectively rendered the area between the wells hydrophobic. HDTwas selected because it is believed to oxidize PDMS, but it does notdissolve in contact with aqueous solution. It is believed that otherreagents may be substituted. The difference in wettability between thewells, which are hydrophilic, and the region between the wells, whichare hydrophobic, can enable easy filling and confinement of a aqueoussolution containing a biomolecule of interest to the microwells during afilling step. As shown in FIG. 24D, the microwell 320 can then be filledwith a solution 330.

Specifically, a poly(ethylene terephthalate) (PET) film (obtained fromDupont, Inc.) was chemically derivatized to introduce COOH groups. Yang,Z. P.; Chilkoti, A. Adv. Mater, 2000, 12, 413. The surface COOH groupswere then reacted with N-hydroxysuccinimide (NHS) (0.2 M) and1-ethyl-3-(dimethyl-amino)propyl carbodiimide (EDAC) (0.1 M) indistilled water. The NHS-ester-functionalized surface was dried undernitrogen and used immediately for micropatterning biomolecules.

A cell-adhesive peptide was covalently patterned using microwelltechniques. Massia, S. P.; Hubbell, J. A. J. Biomed. Mater. Res. 1991,25, 223.; Rezania, A.; Thomas, C. H.; Branger, A. B.; Waters, C. M.;Healy, K. W. J. Biomed. Mater. Res. 1997, 37, 9. In certain embodiments,biotin can be patterned on a surface and then a steptavidin-biotinsystem can be used subsequently to pattern a biotinylated peptide.Wilchek, M.; Bayer, E. A. Avidin-Bioltin Technology; Methods inEnzymology, Vol. 184; Academic Press: Sandiego, Calif., 1990; Green, N.M. Biochem J. 1966, 101, 774. In other embodiments, direct, covalentpatterning of the peptide can be performed onto a derivatized PETsurface. Both biotin-GRGDSP(K-TMR)-NH₂ were synthesized by (lower case)Anaspec, Inc. (San Jose, Calif.) and were of over 90% purity.Tetramethyl rhodamine (TMR) was covalently attached to the amine moietyin the lys (K) residue during solid-phase synthesis of each peptide.

In separate experiments, 100 μL of a 10 mM solution of EZ-Linkbiotin-PEO-LC-amine, which is a long-chain (LC) derivative of biotinwith a poly-(ethylene oxide) (PEOP) spacer (22.0 Å spacer length),((+)-biotinyl-3,6,9-trioxaundecanediamine, Pierce) (biotin-NH₂) inphosphate buffer or gly-arg-gly-asp-ser-pro-lys)tetramethylrhodamine)NH₂in phosphate buffer (GRGDSP(K-TMR)NH₂) were pipetted onto the PDMSmicrowells 320, and the droplets that remained on the hydrophobicsurface between the microwells were blown away with a stream of nitrogengas. The derivatized polymer surface 340 was then brought into physicalcontact with the PDMS microwells 320 as shown in FIG. 24E–F. Theelastomeric nature of PDMS can provide a substantially leak-proof sealbetween the microwell 320 and the surface 340. The entire assembly wasthen inverted, as shown in FIG. 24F in order to bring the solution 330in contact with the surface 340. The reaction between biotin-NH₂ (orGRGDSP(K-TMR)-NH₂) and the NHS esters on the surface of PET was allowedto proceed for 30 minutes at room temperature. Subsequently, the surface340 was lifted away from the microwell 320 and extensively washed inethanol, leaving the ligand 350 on the the surface 340.

After patterning biotin-NH₂ on PET using the process described herein,the surface was incubated with 0.1 μM unlabeled streptavidin orAlexa488-labeled streptavidin in 10 mM HEPES, 0.02%(v/v) Tween 20detergent for 30 minutes at room temperature. The samples were washedexhaustively in buffer, and confocal fluorescence microscopy was used toexamine each stage of the patterning of the biotinylated peptide.Spatially well-resolved patterns of streptavidin were observed (FIG.26A) with a signal-to-noise ration (S/N) in the patterns (patternintensity/background intensity) of 10±0.9 (FIG. 26B), which is greaterthan the S/N of about 5 that is typically obtained by reactive μCP onPET. A control experiment was performed by incubating a biotinmicropattern with Alexa488-labeled streptavidin that had beenpresaturated with free biotin. No patterns were observed by fluorescencemicroscopy. Without intending to be bound by theory, it is believed thatthe formation of streptavidin patterns was caused by molecularrecognition between the protein and micropatterned biotin on thesurface. It is also believed that a higher contrast of streptavidinpatterns obtained by the microwell patterns compared to μCP ofbiotin-NH₂ on PET (see Yang, Z. P.; Chilkoti, A. Adv. Mater. 2000, 12,413. and Yang, Z. P.; Belu A. M.; Liebmann-Vinson, A.; Sugg, H.;Chilkoti, A. Langmuir 2000, 16, 7482.) may result from the longercontact time of the surface with biotin-NH₂ than is typically possiblewith μCP, thereby providing a higher surface density of immobilizedbiotin.

Next, an unlabled streptavidin pattern was incubated with 0.1 μMbiotin-GRGDSP(K-TMR) (10 mM HEPES, 0.02%(v/v) Tween 20 detergent)resulting in the formation of a peptide micropattern (FIG. 27A). The S/Nin the pattern was 7±0.5 (FIG. 27B). The feasibility of direct micropatterning of a GRGDSPK(TMR)-NH₂ peptide was also investigated byreaction between the NH₂ moiety and the NHS ester on the surface (FIG.28A). Patterning was successful, and a S/N of the peptide patternscreated by such covalent coupling was 5±0.8 (FIG. 28B). It is noted thatthe S/N of the peptide patterns created by covalent coupling (FIG. 28B)was lower than the S/N of 7±0.5 (FIG. 27B) (P<0.001, unpaired t-test)obtained for its biotin-conjugate patterned using thestreptavidin-biotin system. Without wishing to be bound by theory, it isbelieved that the lower S/N obtained by covalent patterning is due tothe limited reactivity of the amine moiety which is caused by sterichindrance from the adjacent dye molecule.

The processes described in FIGS. 24A–G and the accompanying discuss maybe performed on a variety of surface materials, including polymers, SAMson gold or glass. The concentration of the biomolecule in the aqueoussolution and its contact time with the surface can be varied over a widerange. Low-molecular weight PDMS is generally not left behind in thepatterned regions as has been observed with μCP methods. Isolatedstructures such as circles, squares, stripes and other patterns can befabricated. Microwell patterning methods can be interfaced withpiezoelectric or inkjet dispensers to simultaneously create patterns ofmany different biomolecules. Thus, spatial resolution may be dictated bythe size of the microwells, which can be controlled, and not by dropletspreading.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of attaching a ligand to a surface, comprising: contacting asurface with a substrate containing an amphiphilic comb polymer whereinthe substrate is configured to provide a pattern of the amphiphilic combpolymer on a selected region of the surface; separating the substratefrom the surface, thereby leaving the amphiphilic comb polymer on theselected region of the surface to provide a selected region of thesurface having amphiphilic comb polymer thereon; and then depositing aligand on the surface such that the selected region of the surfacehaving the amphiphilic comb polymer thereon is substantially free of theligand.
 2. The method of claim 1, wherein the surface is a polymersurface.
 3. The method of claim 1, wherein the ligand is a biologicalligand.
 4. The method of claim 1, wherein the substrate comprises astamp, and wherein the amphiphilic comb polymer is attached to a surfaceof the stamp.
 5. The method of claim 1, wherein the substrate comprisesat least one well and wherein an aqueous solution is present in the atleast one well, the aqueous solution comprising the amphiphilic combpolymer.
 6. The method of claim 1, wherein the ligand is deposited byadsorption from a solution.
 7. The method of claim 1, wherein the ligandis deposited by chemical conjugation from a solution.
 8. The method ofclaim 1, wherein the amphiphilic comb polymer comprises a backboneformed of a hydrophobic water-insoluble polymer and at least one sidechain formed of a hydrophilic polymer.
 9. The method of claim 8, whereinthe hydrophobic water-insoluble polymer comprises a biodegradablepolymer.
 10. The method of claim 9, wherein the biodegradable polymer isselected from the group consisting of poly(amino acids),poly(anhydrides), poly(orthoesters), poly(phosphoesters), polylactones,poly(sebacate), poly(hydroxy acids), copolymers thereof, and mixturesthereof.
 11. The method of claim 8, wherein the hydrophobicwater-insoluble polymer comprises a non-biodegradable polymer.
 12. Themethod of claim 11, wherein the non-biodegradable polymer is selectedfrom the group consisting of polyalkylenes, polyvinyl ethers, polyvinylesters, polysiloxanes, polystyrene, polyurethanes, polyacrylates,polyacrylamides, copolymers thereof, and mixtures thereof.
 13. Themethod of claim 8, wherein the hydrophilic polymer is formed frompolymeric blocks selected from the group consisting of poly(ethyleneglycol), poly(ethylene oxide), poly(propylene glycol), poly(propyleneoxide), partially or fully hydrolyzed poly(vinyl alcohol),poly(vinylpyrrolidone), dextran, and mixtures thereof.
 14. The method ofclaim 1, wherein the ligand is selected from the group consisting ofsmall biological molecules, proteins, peptides, nucleic acids, lipids,saccharides, oligosaccharides, carbohydrates, lipopolysaccharide,lipoprotein, peptide nucleic acids (PNA), ribozymes, DNA or PNA aptamer.15. The method of claim 1, wherein the ligand is a small biologicalmolecule.
 16. The method of claim 1, wherein the ligand is a peptide.17. The method of claim 1, wherein the ligand is a protein.
 18. Themethod of claim 1, wherein the ligand is biotin.
 19. The method of claim1, wherein the ligand is a synthetic polymer.
 20. The method of claim 1,wherein the ligand is a biological polymer.
 21. The method of claim 1,wherein the surface is the surface of a polymer selected from the groupconsisting of poly(ethylene terephthalate) (PET), polystyrene (PS),polycarbonate (PC), poly(epsilon-caprolactone) (PECL or PCL),poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA),polydimethylsiloxane (PDMS), polybutadiene (PB), polyvinylalcohol (PVA),fluorinated polyacrylate (PFOA), poly(ethylene-butylene) (PEB),poly(tetrafluoroethylene), and poly(styrene-acrylonitrile) (SAN). 22.The method of claim 1, wherein the surface is configured as a flatsurface.
 23. The method of claim 1, wherein the surface is configured asa curved surface.
 24. The method of claim 4, wherein the stamp is anelastomeric stamp.
 25. The method of claim 4, wherein the stamp is apoly(dimethylsiloxane) (PDMS) stamp.
 26. The method of claim 4, whereinthe stamp is plasma-oxidized prior to the contacting step.
 27. Themethod of claim 4, wherein the stamp is chemically oxidized prior to thecontacting step.
 28. The method of claim 1, wherein the ligand iscytophilic.
 29. The method of claim 1, further comprising bindinganother ligand to the at least one ligand on the surface after theseparating step.
 30. The method of claim 29, wherein the other ligand isstreptavidin, and the ligand covalently bound to the surface after theseparating step is biotin.